EA039429B1 - Methods for tumor treatment using cd3cd20 bispecific antibody - Google Patents

Abstract

The invention relates to a method (dosage regimen) for administering a CD3CD20 bispecific antibody to a human patient, comprising (a) administering a first dose of said antibody in a specific dosage; and consecutively (b) administering a second dose of said antibody after a period of time, wherein said second dose exceeds said first dose. The methods of the invention (and likewise the dosage regimen of the invention) are also suitable for treating B cell (CD20-positive) cancer in a human patient, or for ameliorating and/or preventing a medical condition mediated by the periodic or continued administration of a CD3CD20 bispecific antibody to a human patient. The invention also relates to the use of a CD3CD20 bispecific antibody for the preparation of a pharmaceutical composition to be used in a method or treatment regime as defined in any one of the preceding claims. A pharmaceutical package or kit comprising the first dose, the second dose, and any subsequent doses are also part of the invention.

Description

(57) The invention relates to a method (dosing schedule) for administering a CD3xCD20 bispecific antibody to a human patient, comprising: (a) administering a first dose of said antibody at a specific dosage and thereafter (b) administering a second dose of said antibody after a certain period of time, wherein said the second dose exceeds the indicated first dose. The methods of the invention (and likewise the dosing regimens of the invention) are also suitable for treating B-cell (CO20 positive) cancer in a human patient, or for ameliorating and/or preventing the condition by intermittent or continuous administration of a CD3xCD20 bispecific antibody to a patient 039429 B1 to a person. The present invention also relates to the use of a bispecific antibody

CD3xCD20 for the preparation of a pharmaceutical composition for use in the method or treatment regimen specified in any of the preceding paragraphs. The pharmaceutical package or kit containing the first dose, the second dose and any subsequent doses are also part of the present invention.

Cross-reference to related applications

This application claims priority in US Provisional Application No. 62/080716 filed November 2014 and US Provisional Application No. 62/160788 filed May 13, 2015, the entire contents of which are incorporated herein by reference.

Sequence listing

This application contains the Sequence Listing filed in machine-readable format as a file called 10162WO01_ST25.txt created on June 3, 2014 (83392 bytes).

Technical field

The present invention relates to bispecific antibodies targeting CD20 and CD3 antigens and methods for killing tumors. Also, the present invention relates to methods for reducing and/or regulating effector functions, which can be achieved as a result of Fc binding in combination with antibody therapy for the treatment of tumors.

State of the art

A bispecific antibody having a CD20 binding arm and a CD3 binding arm may provide the necessary cross-talk to enhance antitumor activity. The third modality of such a bispecific antibody is the Fc domain. It has been found that modification of the Fc-binding properties enhances the antitumor efficacy of the therapeutic antibody.

Binding of the Fc domain of an immunoglobulin to its receptors leads effector cells to sites of bound antigen, which ultimately leads to a variety of signaling and immune responses. These diverse effector functions, such as CDC and ADCC, are the result of class G immunoglobulins (IgG) complexing between the IgG Fab domain and the target antigen, with the IgG Fc domain binding to Fc receptors on effector cells. Some effector functions of IgG are independent of antigen binding and include functions such as circulating serum levels and the ability to transport Ig across barriers. Other effector functions are considered important for the use of immunoglobulin therapy such as cancer treatment. In particular, the ADCC mechanism is considered to be one of the main antitumor mechanisms of therapeutic antibodies already on the market, such as rastuzumab (metastatic breast cancer) and rituximab (non-Hodgkin's lymphoma).

Current therapeutic strategies generally suggest that reducing effector functions (or reducing Fc-gamma receptor binding) by modified antibody Fc domains may be beneficial for antibodies that aim to neutralize or inhibit the biological activity of an antigen (for example, antibodies that block or antagonists) or activation or initiation of subsequent cell signaling (eg, antibody agonists).

However, the development of tumor-targeted antibodies with reduced effector function is contrary to the concept of tumor therapy, since reduced cytotoxicity (i.e., ADCC and CDC) of target cells is not expected to be effective for treating the disease, i.e. destruction of tumor cells or inhibition of tumor growth.

One of the strategies described herein uses differential Fc receptor binding in combination with bispecific antigen binding to specifically target tumor markers as well as trigger tumor-specific killing of T cells. The Fc domain of an antibody is designed to carefully regulate Fc receptor binding to stop or reduce unwanted cell killing, such as T cells, natural killer cells, and Fc receptor-bearing macrophages. The unique binding profile for Fc receptor interactions, including FcγRII receptor binding interactions but not FcyRI or FcyRIII interactions, has surprisingly been beneficial for tumor-targeted Ig therapy in the context of bispecific antibodies that bind both CD3 and CD20. However, there is still a need to find new therapies that stimulate the immune system and are effective in ablation of tumors, without leading to excessive release of cytokines and toxicity to the patient.

Current bispecific therapies such as BiTE® antibodies (bispecific T-cell activator) are administered in extremely small doses, but at frequent intervals. There is an unmet medical need for additional treatment options with tolerated dosing regimens for patients with B-cell CD20+ malignancies, especially for patients who relapse or progress after initial therapy.

The essence of the invention

In a first aspect, the present invention provides bispecific antibodies with altered Fc binding domains that bind human CD3 and CD20 and are further engineered to have specific effector functions that are not found in the natural repertoire of the immune system. The antibodies of this aspect of the invention are useful, among other things, for targeting CD3-expressing T cells and for stimulating

- 1 039429 T cell activation, for example under conditions where T cell mediated killing is beneficial or desirable as part of the action of a bispecific antibody that directs CD3 mediated T cell activation to certain cell types, such as tumor cells with CD20 antigen. The antibodies of the invention are administered according to a dose escalation protocol to enhance their effectiveness in stimulating the immune system, i.e. T cell activation while minimizing toxic effects such as cytokine storm.

In another aspect, the invention provides the use of a bispecific antibody for treating or ameliorating B-cell cancer, or a method of treating a subject, comprising administering a first dose of an antibody over a first time period and sequentially administering a second dose of said antibody over a second time period, said second dose exceeds said first dose, and the bispecific antibody comprises a first antigen-binding domain that binds human CD3, a second antigen-binding domain that binds human CD20, and a chimeric Fc domain associated with each of the first and second antigen-binding domains, and treating or ameliorating cancer comprises : (a) inhibition of tumor growth in the subject, (b) mediation of B cell lysis in the subject, (c) treatment of B cell cancer in the subject, (d) treatment of a CD20-positive cancer in the subject, or (e ) treatment of CD20-expressing melanoma cancer in a subject.

In another aspect, the invention provides the use of a bispecific antibody to treat or reduce tumor burden or cancer, or a method of treating a subject, comprising administering a first dose of the antibody over a first period of time and sequentially administering a second dose of said antibody over a second period of time, said second dose being greater than said first dose, and the bispecific antibody comprises a first antigen-binding domain that binds human CD3, a second antigen-binding domain that binds a human tumor target antigen or tumor-specific antigen, and a chimeric Fc domain associated with each of the first and second antigen-binding domains, and treatment or reducing tumor burden or cancer includes: (a) inhibiting tumor growth in a subject, (b) mediating tumor cell lysis in a subject, (c) treating cancer positive for expression of a tumor target antigen or tumor-specific antigen in a subject kta or (d) treating a tumor target antigen-expressing cancer or tumor-specific antigen-expressing tumors in a subject.

Typical anti-CD3/CD20 antibodies according to the present invention are listed in table. 1-8 in this document. In table. 1 shows the amino acid sequence designations of the heavy chain variable regions (HCVR) and light chain variable regions (LCVR), as well as the heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and the light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) typical bispecific antibodies. In table. 2 shows the sequence designations of nucleic acid molecules encoding HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 generic bispecific antibodies. In table. 3 shows the designations for amino acid sequence combinations of exemplary bispecific antibodies, including combinations of HCVR, heavy chain constant region (CH), and LCVR. In table. 4 shows the nucleotide sequence designations for combinations of nucleic acid molecules encoding combinations of HCVR, heavy chain constant region (CH), and LCVR of exemplary bispecific antibodies.

In table. 5 describes the designations of amino acid sequences for examples of the heavy chain according to the invention, while the bispecific antibody contains HCVR containing HCDR1, HCDR2 and HCDR3 from table. 5 paired with CH according to the invention. In table. 6 separately describes the designations of amino acid sequences for light chain examples according to the invention, while the bispecific antibody contains an LCVR containing LCDR1, LCDR2 and LCDR3 from table. 6.

The present invention provides antibodies or antigen-binding fragments thereof containing HCVR containing an amino acid sequence selected from the HCVR amino acid sequences shown in table. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also provides antibodies or antigen-binding fragments thereof containing an LCVR containing an amino acid sequence selected from the LCVR amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also provides antibodies or antigen-binding fragments thereof, containing a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), containing a pair of amino acid sequences from the typical anti-CD3/CD20 antibodies shown in table. 2. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 2/10 (eg, Antibody 1 and Antibody 2).

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a CDR1 heavy chain (HCDR1) comprising an amino acid sequence selected from the HCDR1 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides antibodies or antigen-binding fragments thereof containing a CDR2 heavy chain (HCDR2) containing an amino acid sequence selected from the HCDR2 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides antibodies or antigen-binding fragments thereof containing a CDR3 heavy chain (HCDR3) containing an amino acid sequence selected from the HCDR3 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a CDR1 light chain (LCDR1) containing an amino acid sequence selected from the LCDR1 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides antibodies or antigen-binding fragments thereof containing a CDR2 light chain (LCDR2) containing an amino acid sequence selected from the LCDR2 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides antibodies or antigen-binding fragments thereof containing a CDR3 light chain (LCDR3) containing an amino acid sequence selected from the LCDR3 amino acid sequences shown in Table 1. 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides antibodies or antigen-binding fragments thereof, containing a pair of HCDR3 and LCDR3 amino acid sequences (HCDR3/LCDR3) containing the HCDR3 amino acid sequences shown in Table 1. 1, paired with any of the LCDR3 amino acid sequences shown in table. 1 such that the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8/16 (eg, Antibody 1 or Antibody 2).

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a group of six CDRs (ie, HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) shown in Table 1. 1. In certain embodiments of the invention, the amino acid sequence group HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is represented by SEQ ID NO: 4-6-8-20-22-24; or 1214-16-20-22-24 (eg Antibody 1 and Antibody 2).

In a related embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a group of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3) from the HCVR/LCVR amino acid sequence pair indicated for the exemplary antibodies shown in Table . 1. For example, included in the present invention are antibodies or antigen-binding fragments thereof comprising a group of amino acid sequences HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 from a pair of amino acid sequences HCVR/LCVR selected from the group consisting of SEQ ID NO: 2/18 (for example , Antibody 1 and Antibody 2). Methods and techniques for determining CDRs within HCVR and LCVR amino acid sequences are well known in the art and applicable to determining CDRs within specific HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary techniques that can be used to define CDR boundaries include, for example, the Kabat definition, the Hotia definition, and the AbM definition. In general terms, Kabat's definition is based on sequence variability, Hotia's definition is based on the location of structural loop regions, and AbM's definition is a compromise between Kabat's and Hotia's approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); AlLazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. sci. USA 86:9268-9272 (1989). Public databases are also available for determining antibody CDR sequences.

The present invention also provides nucleic acid molecules encoding antibodies or parts thereof, for example, the present invention provides nucleic acid molecules encoding the HCVR or LCVR amino acid sequences shown in Table 1. one; in certain embodiments of the invention, the nucleic acid molecule contains a polynucleotide sequence selected from the HCVR/LCVR nucleotide sequences shown in table. 2,

- 3 039429 or substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with them.

The present invention also provides nucleic acid molecules encoding the amino acid sequences of HCDR1 or HCDR2 or HCDR3 shown in table. one; in certain embodiments of the invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the nucleotide sequences HCDR1 or HCDR2 or HCDR3 shown in table. 2, or substantially similar in sequence, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also provides nucleic acid molecules encoding any of the LCDR1 or LCDR2 or LCDR3 amino acid sequences shown in Table 1. one; in certain embodiments of the invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the LCDR1 or LCDR2 or LCDR3 nucleotide sequences shown in Table. 2, or substantially similar in sequence, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them.

The present invention also provides nucleic acid molecules encoding HCVR, wherein the HCVR comprises a group of three CDRs (i.e., HCDR1-HCDR2-HCDR3) wherein the group of amino acid sequences HCDR1-HCDR2-HCDR3 is as specified for the exemplary anti-CD3 antibodies shown in table. one.

The present invention also provides nucleic acid molecules encoding an LCVR, wherein the LCVR contains a group of three CDRs (i.e. LCDR1-LCDR2-LCDR3) wherein the LCDR1-LCDR2-LCDR3 amino acid sequence group is as specified for the exemplary anti-CD3 antibodies shown in table. one.

The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the HCVR contains an amino acid sequence from the HCVR amino acid sequences shown in Table 1. 1, a LCVR contains the amino acid sequence of the LCVR amino acid sequences shown in table. 1. In certain embodiments of the invention, the nucleic acid molecule contains a polynucleotide sequence selected from any of the HCVR nucleotide sequences shown in table. 2 or substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them, and a polynucleotide sequence selected from the LCVR nucleotide sequences given in tab. 2, or substantially similar in sequence, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with them. In certain embodiments in accordance with this aspect of the invention, the nucleic acid molecule encodes HCVR and LCVR, and HCVR and LCVR are derived from a single anti-CD3 antibody, shown in table. one.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain constant region (CH) comprising an amino acid sequence selected from the CH amino acid sequences shown in Table 1. 2, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a heavy chain constant region (CH) encoded by a nucleotide sequence selected from the CH nucleotide sequences shown in Table 1. 2, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides recombinant expression vectors capable of expressing a polypeptide comprising an anti-CD3 antibody heavy or light chain variable region and an anti-CD20 antibody heavy or light chain variable region. For example, the present invention includes recombinant expression vectors containing any of the above nucleic acid molecules, i.e. nucleic acid molecules encoding any of the sequences HCVR, LCVR and/or CDR and/or CH sequences shown in table. 1 and 2. Also included within the scope of the present invention are host cells in which such vectors have been introduced, as well as methods for producing antibodies or parts thereof by culturing the host cells under conditions that produce antibodies or antibody fragments and isolating the antibodies thus obtained. or antibody fragments.

In another aspect, the invention provides a therapeutically effective amount of a recombinant human antibody or fragment thereof that specifically binds CD3 and CD20, the antibody comprising a chimeric Fc domain, and a pharmaceutically acceptable carrier. In related

- 4 039429 aspect, the invention relates to a composition which is a combination of an aHTu-CD3/CD20 antibody and a second therapeutic agent. In one embodiment of the invention, the second therapeutic agent is any agent that provides an advantage in combination with an anti-CD3/CD20 antibody. Exemplary agents that may provide benefit in combination with an anti-CD3/CD20 antibody include, without limitation, other agents that bind and/or activate CD3 signaling (including other antibodies or antigen-binding fragments thereof, etc.), and/ or other agents that do not directly bind CD3 but nonetheless activate or stimulate immune cell activation or enhance tumor killing. Additional combination therapy options and co-formulations comprising anti-CD3 antibodies of the present invention are disclosed elsewhere in this document.

According to another aspect, the present invention provides bispecific antigen-binding molecules that bind CD3 and a target antigen, the molecule comprising a chimeric Fc domain having reduced effector function. In a specific embodiment of the invention, the molecule contains the chimeric Fc domain described herein. In accordance with certain exemplary embodiments of the invention, the bispecific antigen-binding molecules bind CD3 and CD20; such bispecific antigen-binding molecules are also referred to herein as anti-CD3/anti-CD20 bispecific molecules. The Ahtu-CD20 portion of the anti-CD3/anti-CD20 bispecific molecule is useful for targeting tumor cells that express CD20 (eg, B cell tumors), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. The simultaneous binding of CD20 on the tumor cell and CD3 on the T cell mediates targeted killing (cell lysis) of the target tumor cell by the activated T cell, which is facilitated by effector cells that bind the chimeric Fc domain. Therefore, the anti-CD3/anti-CD20 bispecific molecules of the invention are useful, among other things, in the treatment of diseases or disorders associated with or caused by CD20-expressing tumors (eg, lymphomas and melanoma tumors).

The anti-CD3/anti-CD20 bispecific molecules of the invention further provide a method for achieving regression of CD20 positive tumors. Therefore, the invention provides a method of treating B-cell cancer in a subject, comprising administering a therapeutic amount of anti-CD3/anti-CD20 bispecific molecules according to the invention, said amount being sufficient to reduce tumor burden, achieve tumor regression, or reduce tumor development in the subject.

The anti-CD3/anti-CD20 bispecific molecules of the invention further provide a method for suppressing or achieving regression of CD20-positive melanoma. Therefore, the invention provides a method of treating melanoma in a subject, comprising administering a therapeutic amount of anti-CD3/anti-CD20 bispecific molecules according to the invention, said amount being sufficient to inhibit tumor growth, reduce tumor burden, or reduce tumor development in the subject.

The bispecific antigen-binding molecules according to this aspect of the present invention comprise a first antigen-binding domain that specifically binds human CD3 and a second antigen-binding domain that specifically binds CD20 and a chimeric Fc domain. The present invention includes anti-CD3/anti-CD20 bispecific molecules (eg, bispecific antibodies) wherein each antigen binding domain comprises a heavy chain variable region (HCVR) coupled to a light chain variable region (LCVR). In certain exemplary embodiments, the anti-CD3 antigen-binding domain and the anti-CD20 antigen-binding domain comprise different, distinct HCVRs coupled to a common LCVR. For example, as illustrated in Example 2 of this document, bispecific antibodies were constructed comprising a first antigen-binding domain that specifically binds CD3, the first antigen-binding domain comprising an HCVR/LCVR pair derived from an anti-CD3 antibody; and a second antigen-binding domain that specifically binds CD20, wherein the second antigen-binding domain comprises an HCVR derived from an anti-CD20 antibody coupled to an LCVR derived from an anti-CD3 antibody (e.g., the same LCVR that is part of the anti-CD3 antigen-binding domain ). In other words, in exemplary molecules disclosed herein, coupling an HCVR from an anti-CD20 antibody to an LCVR from an anti-CD3 antibody results in an antigen-binding domain that specifically binds CD20 (but does not bind CD3). In such embodiments, the first and second antigen-binding domains contain different anti-CD3 and anti-CD20 HCVR, but share a common anti-CD3 LCVR.

The present invention provides anti-CD3/anti-CD20 bispecific molecules in which the first antigen-binding domain that specifically binds CD3 contains any of the HCVR amino acid sequences shown in Table 1. 1 or 2. The first antigen-binding domain that specifically binds CD3 may also contain any of the LCVR amino acid sequences shown in table. 1 or 2. In accordance with certain embodiments of the invention, the first antigen-binding domain that specifically binds CD3,

- 5 039429 contains any of the pairs of amino acid sequences HCVR/LCVR shown in table. 1 or 2. The present invention also provides anti-CD3/anti-CD20 bispecific molecules, wherein the first antigen-binding domain that specifically binds CD3 comprises any of the heavy chain CDR1-CDR2-CDR3 amino acid sequences shown in Table 1. 1 or 2, and/or any of the light chain CDR1-CDR2-CDR3 amino acid sequences shown in Table. 1 or 2.

In accordance with certain embodiments, the present invention provides anti-CD3/anti-CD20 bispecific molecules wherein the first antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region (HCVR) having an amino acid sequence comprising SEQ ID NO: 10 , or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-CD20 bispecific molecules, wherein the first antigen-binding domain that specifically binds CD3 comprises a light chain variable region (LCVR) having an amino acid sequence comprising SEQ ID NO: 18, or to a significant extent a sequence similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-CD20 bispecific molecules in which the first antigen-binding domain that specifically binds CD3 contains a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR) selected from the group consisting of SEQ ID NO: 10 /eighteen.

The present invention also provides anti-CD3/anti-CD20 bispecific molecules, wherein the first antigen-binding domain that specifically binds CD3 comprises a heavy chain CDR3 domain (HCDR3) having an amino acid sequence comprising SEQ ID NO: 16, or to a significant extent a sequence similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 domain (LCDR3) having an amino acid sequence comprising SEQ ID NO: 24, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least least 99% sequence identity.

In certain embodiments of the invention, the first antigen-binding domain that specifically binds CD3 contains a pair of amino acid sequences HCDR3/LCDR3 containing SEQ ID NO: 16/24.

The present invention also provides anti-CD3/anti-CD20 bispecific antigen-binding molecules, wherein the first antigen-binding domain that specifically binds CD3 comprises a heavy chain CDR1 domain (HCDR1) having an amino acid sequence comprising SEQ ID NO: 12, or substantially a sequence similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 domain (HCDR2) having an amino acid sequence comprising SEQ ID NO: 14, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a light chain CDR1 domain (LCDR1) having an amino acid sequence comprising SEQ ID NO: 20, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR2 domain (LCDR2) having an amino acid sequence comprising SEQ ID NO: 22, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least least 99% sequence identity.

Certain non-limiting exemplary anti-CD3/anti-CD20 bispecific antigen-binding molecules of the invention comprise a first antigen-binding domain that specifically binds CD3, comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively having amino acid sequences selected from the group consisting of SEQ ID NO: 12-14-16-20-22-24.

The present invention also provides anti-CD3/anti-CD20 bispecific molecules wherein the second antigen-binding domain that specifically binds CD20 comprises a heavy chain variable region (HCVR) having an amino acid sequence comprising SEQ ID NO: 2, or to a significant extent a sequence similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-CD20 bispecific molecules in which the second antigen-binding domain that specifically binds CD20 contains the variant

- 6 039429 white light chain region (LCVR) having an amino acid sequence containing SEQ ID NO: 18 or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. The present invention also provides anti-CD3/anti-CD20 bispecific molecules wherein the second antigen-binding domain that specifically binds CD20 comprises a light chain variable region (LCVR) having an amino acid sequence comprising SEQ ID NO: 75, or to a significant extent a sequence similar to it having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also provides anti-CD3/anti-CD20 bispecific molecules, in which the second antigen-binding domain that specifically binds CD20 contains a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR) containing SEQ ID NO: 2/18 or SEQ ID NO: 2/75.

The present invention also provides anti-CD3/anti-CD20 bispecific molecules wherein the second antigen-binding domain that specifically binds CD20 comprises a heavy chain CDR3 domain (HCDR3) having the amino acid sequence of SEQ ID NO: 8, or substantially similar to her sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a light chain CDR3 domain (LCDR3) having an amino acid sequence comprising SEQ ID NO: 24, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least least 99% sequence identity.

In certain embodiments of the invention, the second antigen-binding domain that specifically binds CD20 contains a pair of amino acid sequences HCDR3/LCDR3 containing SEQ ID NO: 8/24.

The present invention also provides anti-CD3/anti-CD20 bispecific antigen-binding molecules, wherein the second antigen-binding domain that specifically binds CD20 comprises a heavy chain CDR1 domain (HCDR1) having the amino acid sequence of SEQ ID NO: 4, or substantially similar to with it a sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 domain (HCDR2) having the amino acid sequence of SEQ ID NO: 6, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identities; a light chain CDR1 domain (LCDR1) having the amino acid sequence of SEQ ID NO: 20, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identities; and a light chain CDR2 domain (LCDR2) having the amino acid sequence of SEQ ID NO: 22, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least 99 % sequence identity.

Certain non-limiting exemplary anti-CD3/anti-CD20 bispecific antigen-binding molecules of the invention comprise a second antigen-binding domain that specifically binds CD20, comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively having amino acid sequences selected from the group consisting of SEQ ID NO: 4-6-8-20-22-24 (eg Antibody 1 and Antibody 2).

In a related embodiment, the invention includes anti-CD3/anti-CD20 bispecific antigen-binding molecules, wherein the second antigen-binding domain that specifically binds CD20 comprises the heavy and light chain CDR domains contained in the heavy and light chain variable region sequences (HCVR/LCVR) SEQ ID NO: 2/18.

In another aspect, the present invention provides nucleic acid molecules encoding any of the HCVR, LCVR, or CDR sequences of the anti-CD3/antiCD20 bispecific antigen-binding molecules disclosed herein, including nucleic acid molecules containing the polynucleotide sequences shown in Table 1. 2, 7 and 8, as well as nucleic acid molecules containing two polynucleotide sequences shown in table. 2, 7 and 8, in any functional combination or order. Also included in the invention are recombinant expression vectors carrying the nucleic acids of the invention and host cells into which such vectors have been introduced, as well as methods for producing antibodies by culturing the host cells under antibody-producing conditions and isolating the resulting antibodies.

The present invention includes anti-CD3/anti-CD20 bispecific antigen-binding molecules, in which any of the above antigen-binding domains that specifically bind CD3 is combined, fused or otherwise associated with any of the above antigen-binding domains that specifically bind CD20, to bispecial education

- 7 039429 physical antigen-binding molecule that binds CD3 and CD20.

In another aspect, the invention provides a bispecific antibody comprising a first antigen-binding domain that binds human CD3, a second antigen-binding domain that binds human CD20, and a chimeric Fc domain associated with each of the first and second antigen-binding domains. In a related aspect, the bispecific antibody is capable of specifically binding to human FcyRIIA and human FcyRIIB. The present invention provides anti-CD3/anti-CD20 bispecific antigen-binding molecules that preferentially bind to human FcyRIIA and human FcyRIIB and exhibit little or no binding affinity for human FcyRI or human FcyRIII. The bispecific antibodies of the invention are able to specifically bind to human FcyRIIA and human FcyRIIB with a higher affinity than these antibodies bind to human FcyRI or human FcyRIII, according to in vitro assay data. In some embodiments, the antibody specifically binds to both human FcyRIIA and human FcyRIIB and exhibits a binding affinity of less than 1 μM Kd for each of human FcyRI and human FcyRIII as determined by an in vitro assay.

In other aspects, the invention provides a bispecific antibody comprising a first and second heavy chain polypeptide each containing a chimeric Fc domain, wherein the first heavy chain polypeptide comprises an antigen-binding domain that binds human CD3 and the second heavy chain polypeptide comprises a second antigen-binding domain that binds human CD20.

In other embodiments, the antibody exhibits greater binding affinity for human FcyRIIA compared to human FcyRIIB, as determined by in vitro assay data. In other embodiments, the antibody binds to human FcyRIIA and exhibits a lower CD compared to human FcyRIIB binding as determined by an in vitro assay. In some embodiments of the invention, the antibody binds to human FcyRIIA at 25°C with a Kd value of 10 to 30 μM according to in vitro assay data. In some embodiments of the invention, the antibody binds to human FcyRIIB at 25°C with a Kd value between 100 and 250 μM according to in vitro assay data.

In another embodiment, the antibody exhibits little or no detectable binding affinity for human FcyRI according to in vitro assay data. In some embodiments, the antibody exhibits little or no detectable binding affinity for human FcyRIII as determined by an in vitro assay.

In some embodiments, the in vitro assay is a surface plasmon resonance assay.

In some embodiments, the antibody exhibits reduced antibody-dependent cellular cytotoxicity (ADCC) compared to an antibody containing a wild-type Fc domain, as determined by an in vitro cytotoxicity assay.

In some embodiments, the antibody exhibits negligible or no detectable antibody-dependent cellular cytotoxicity (ADCC).

In some embodiments, the antibody exhibits reduced complement-dependent cytotoxicity (CDC) compared to an antibody containing a wild-type Fc domain, as determined by an in vitro cytotoxicity assay.

In some embodiments, the antibody exhibits less than 50% complement dependent cytotoxicity (CDC) of the total cell population.

In some embodiments of the invention, the antibody exhibits negligible or non-detectable complement-dependent cytotoxicity (CDC).

In some embodiments, the antibody exhibits reduced killing of Fc receptor-bearing cells, such as NK cells or macrophages, compared to an antibody containing a wild-type Fc domain.

In some embodiments, the antibody exhibits reduced killing of Fc receptor-bearing T cells by NK cells or macrophages compared to an antibody containing a wild-type Fc domain.

In some embodiments, the antibody exhibits a binding affinity Kd for human FcyRIIA that is greater than its binding affinity Kd for human FcyRIIB that is greater than its binding affinity Kd for human FcyRI that is greater than or equal to its binding affinity Kd for human FcyRIII, as determined by in vitro data. analysis. In other embodiments, the antibody exhibits a binding affinity Kd for human FcyRIIA > than human FcyRIIB > than human FcyRI > than human FcyRIII as determined by in vitro assay data.

In some embodiments, the antibody exhibits a binding affinity Kd for

- 8 039429 human FcyRIIA that exceeds its binding affinity Kd for human FcyRIIB that exceeds its binding affinity Kd for human FcyRIII that exceeds or equals its binding affinity Kd for human FcyRI according to in vitro assay data. In other embodiments, the antibody exhibits a binding affinity Kd for human FcyRIIA > than human FcyRIIB > than human FcyRIII > than human FcyRI according to in vitro assay data.

In some embodiments, the human FcyRIII is a human FcyRIIIA or a human FcyRIIIB.

In some embodiments, the chimeric Fc domain contains a chimeric hinge region.

In another aspect, the present invention provides a bispecific antibody comprising a first antigen-binding domain, a second antigen-binding domain, and a chimeric heavy chain constant region (CH), wherein (a) the first antigen-binding domain binds CD3, (b) the second antigen-binding domain binds CD20. In certain aspects of the invention, the chimeric CH region binds with greater affinity to human FcyRIIA and human FcyRIIB compared to an antibody containing the wild type CH region, as determined by in vitro assay. In another aspect, the chimeric CH region binds with less or no affinity to human FcyRI and human FcyRIII compared to an antibody containing the wild type CH region, as determined by an in vitro assay.

The invention provides bispecific antibodies containing a chimeric hinge region. In some aspects, the chimeric hinge region contains amino acid sequence residues from positions 216 to 236 (EU numbering). Designed bispecific antibodies according to the invention, in which the chimeric hinge region contains the amino acid sequence of the lower hinge region of human IgG2

PCPAPPVA (SEQ ID NO: 52) 228 to 236 (EU numbering). In certain embodiments, the bispecific antibodies of the invention comprise a chimeric hinge and the upper chimeric hinge comprises amino acid residues 216 to 227 (EU numbering) of the IgG1 upper hinge. In other embodiments, the bispecific antibodies of the invention comprise a chimeric hinge and the upper chimeric hinge comprises amino acid residues 216 to 227 (EU numbering) of the IgG4 upper hinge.

In one embodiment, the bispecific antibody comprises the chimeric hinge amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 53). In another embodiment, the bispecific antibody comprises the chimeric hinge amino acid sequence ESKYGPPCPPCPAPPVA (SEQ ID NO: 54). In certain embodiments of the invention, the bispecific antibody comprises the amino acid sequence of the human IgG4 CH2 domain from positions 237 to 340 (EU numbering). In other embodiments, the bispecific antibody comprises a CH3 domain derived from a human IgG1 CH3 domain or a human IgG4 CH3 domain. In other embodiments, the bispecific antibody comprises a human IgG1 CH1 domain and a human IgG1 CH3 domain. In additional embodiments, the bispecific antibody comprises a human IgG4 CH1 domain and a human IgG4 CH3 domain.

In one aspect, the invention provides a method for producing a bispecific antibody comprising a chimeric heavy chain constant region, comprising: (a) transfecting a host cell with a nucleic acid molecule encoding a first light chain capable of binding the CD3 antigen, said nucleic acid molecule comprising the nucleotide sequence coding for the VL region and a nucleotide sequence encoding the Ig constant CL region, wherein said nucleotide sequence encoding the VL region of the selected antigen-specific antibody and said nucleotide sequence encoding the Ig CL region are operably linked; (b) transfecting the host cell from step (a) with a nucleic acid molecule encoding the first heavy chain of an antibody capable of binding the CD3 antigen, said nucleic acid molecule comprising a nucleotide sequence encoding a VH region and a nucleotide sequence encoding a chimeric constant CH a human Ig region, wherein said nucleotide sequence encoding the VH region and the nucleotide sequence encoding the CH region of said Ig are operably linked; wherein the CH region encodes one or more amino acid modifications in the CH3 domain that reduce or eliminate the binding of the second CH3 domain to protein A; (c) transfecting the host cell from step (a) with a nucleic acid molecule encoding a second heavy chain of an antibody capable of binding the CD20 antigen, said nucleic acid molecule comprising a nucleotide sequence encoding a VH region and a nucleotide sequence encoding a chimeric human CH region. Ig, wherein said nucleotide sequence encoding the VH region and the nucleotide sequence encoding the CH region of said Ig are operably linked; and (c) obtaining said antibody by co-expressing the nucleic acid molecules of (a) and (b) in said host cell.

In some aspects, a method for producing a bispecific antibody optionally comprises transfecting a host cell from step (a) with a nucleic acid molecule encoding a second light chain capable of binding the CD20 antigen, said nucleic acid molecule comprising a nucleotide sequence encoding the VL region of the second light chain, and a nucleotide sequence encoding an Ig constant CL region, wherein said nucleotide sequence encoding a second light chain VL region and said nucleotide sequence encoding an Ig CL region are operably linked.

In some embodiments, the first heavy chain contains a CH3 region containing the H95R modification (according to IMGT exon numbering; H435R according to EU numbering).

In another embodiment, the first heavy chain contains a CH3 region further containing a Y96F (IMGT; Y436F according to EU numbering) modification. In additional embodiments of the invention, the method includes isolating the antibody using protein A.

In another aspect of the invention, a bispecific antibody is provided, comprising: (a) a first heavy chain comprising an antigen-binding domain capable of recognizing and binding a first target antigen, (b) a second heavy chain comprising an antigen-binding domain capable of recognizing and binding a second target antigen, and (c) a common light chain antigen-binding domain capable of recognizing and binding the first or second target antigen, wherein the heavy chain of (a) or (b) or both (a) and (b) comprises a heavy chain constant region (CH) comprising a chimeric hinge region containing the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 54.

In certain embodiments, the heavy chain constant region (CH) comprises the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32. In some embodiments, the chimeric CH-coding nucleotide sequence contains SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO: 33. In other embodiments, the CH chimeric nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32. In other embodiments, the nucleotide sequence of the CH region comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33.

In certain aspects, the bispecific antibody comprises a light chain encoding nucleic acid molecule comprising the amino acid sequence of SEQ ID NO: 35. In other aspects, the bispecific antibody comprises a light chain encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 34.

In certain aspects, the bispecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the amino acid sequence of SEQ ID NO: 37. In other aspects, the bispecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 36.

In certain aspects, the bispecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the amino acid sequence of SEQ ID NO: 40. In other aspects, the bespecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 38 or SEQ ID NO: 39 .

In certain aspects, the bispecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the amino acid sequence of SEQ ID NO: 42. In other aspects, the bispecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 41.

In certain aspects, the bispecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the amino acid sequence of SEQ ID NO: 44. In other aspects, the bispecific antibody comprises a heavy chain-encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 43.

In another aspect, the invention provides a therapeutically effective amount of the anti-CD3/anti-CD20 bispecific antigen binding molecule disclosed herein. In a related aspect, the invention relates to a composition that is a combination of an anti-CD3/anti-CD20 bispecific antigen-binding molecule and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that provides an advantage in combination with an anti-CD3/CD20 bispecific antigen-binding molecule. Exemplary agents that may provide benefit in combination with an antiCD3/CD20 bispecific antigen-binding molecule are discussed in detail elsewhere in this document.

In another aspect, the invention provides therapeutic methods for targeting/killing CD20-expressing tumor cells with anti-CD3/anti- 10 039429

CD20 bispecific antigen binding molecule of the invention, wherein the therapeutic methods comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-CD3/anti-CD20 bispecific antigen binding molecule of the invention. In some embodiments, therapeutic methods for targeting/killing CD20-expressing tumor cells with an anti-CD3/anti-CD20 bispecific antigen-binding molecule or antibody of the invention comprise administering a first dose of the antigen-binding molecule or antibody for a first period of time, and subsequent administration of a second dose of said antibody over a second period of time, said second dose being greater than said first dose.

In some embodiments, the use of a bespecifically antigen-binding molecule comprises administering a first dose of an antibody over a first period of time and then administering a second dose of said antibody over a second period of time, said second dose being greater than said first dose. In certain aspects, the first dose of antibody and the second dose of antibody are in a suitable formulation.

The present invention includes the use of an anti-CD3/anti-CD20 bispecific antigen-binding molecule according to the invention in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by CD20 expression.

The present invention also includes a bispecific antibody comprising a first antigen-binding domain, a second antigen-binding domain and a chimeric heavy chain constant region (CH), wherein: the first antigen-binding domain binds CD3, the second antigen-binding domain binds CD20, the chimeric CH region binds with higher affinity to human FcyRIIA and human FcyRIIB compared to an antibody containing a wild-type CH region, and such a bispecific antibody is intended for use in the manufacture of a medicament. The invention provides a bispecific antibody containing a first antigen-binding domain that binds CD3, a second antigen-binding domain that binds CD20, and a chimeric CH region that binds with higher affinity to human FcyRIIA compared to binding to human FcyRIIB, for use in the manufacture of a medicament .

Other implementation options will become apparent after reading the following detailed description.

Brief description of graphic materials

In FIG. 1 illustrates hinge amino acids used in the construction of chimeric hinge regions and the corresponding amino acid numbering systems.

In FIG. 2 shows the amino acid sequence of the human IgG1 heavy chain constant region, including the CH1, hinge, CH2 and CH3 domains described as IGHG1 in UniProtKB/Swiss-Prot Accn. No. P01857 (SEQ ID NO: 45).

In FIG. 3 shows the amino acid sequence of the human IgG2 heavy chain constant region, including the CH1, hinge, CH2 and CH3 domains described as IGHG2 B UniProtKB/Swiss-Prot Accn. No. P01859 (SEQ ID NO: 46).

In FIG. 4 shows the amino acid sequence of the human IgG4 heavy chain constant region, including the CH1, hinge, CH2 and CH3 domains described as IGHG4 in UniProtKB/Swiss-Prot Accn. No. P01861 (SEQ ID NO: 47).

Fig. 5A and 5B: Dose-response curves illustrating the lack of CSC activity against Daudi (FIG. 5A) and Raji (FIG. 5B) cells in the presence of antibodies having wild type or chimeric CH hinge regions. (Control Antibody 4 = bispecific Ab with C _H IgG1 gt; Antibody 1; Antibody 2; isotype control IgG1 = non-specific Ab with gt IgG1 CH).

Fig. 6A and 6B: Dose-response curves illustrating the lack of ADCC activity against Daudi (FIG. 6A) and Raji (FIG. 6B) cells in the presence of antibodies having wild type or chimeric CH hinge regions. (Control Antibody 4=bispecific Ab with C _H IgG1 gt; Antibody 1; Antibody 2; isotype control IgG1=non-specific Ab with C _H IgG1 gt).

In FIG. 7A-7F illustrate tumor volume (in mm ³ ) over time in NSG mice implanted subcutaneously with a mixture of Raji tumor cells and PBMC (or no PBMC control - FIG. 7D), with the CD3xCD20 bispecific antibody of the invention (Ab 1) either a stock solution or a control antibody was administered after tumor implantation and treatment, starting on the day of tumor implantation (day 0), and measurements were made for 25 days. Fig. 7A: None of the mice showed inhibition of tumor growth when treated with stock solution; fig. 7B: 1 out of 5 (1/5) mice showed tumor growth inhibition when treated with 0.4 mg/kg control Ab5 (antiFelD1 Ab); fig. 7C: 5/5 mice showed tumor growth inhibition when treated with 0.4 mg/kg Antibody 1 (Ab 1); fig. 7D: 0/5 mice showed tumor growth inhibition in the absence of implanted PBMCs and treated with 0.4 mg/kg Ab1; fig. 7E: 5/5 mice showed tumor growth inhibition when treated with 0.04 mg/kg Ab1 and FIG. 7F: 5/5 mice showed tumor growth inhibition when treated with 0.004 mg/kg Ab1.

- 11 039429

In FIG. 8A and 8B illustrate tumor volume (in mm ³ ) over time in NSG mice implanted subcutaneously with a mixture of Raji tumor cells and PBMC and treated with Ab1 (CD3xCD20 chimeric Fc), compared to controls, with or without IgG replenishment (FIG. 8A), or treated with Ab4 (CD3xCD20-dt Fc) versus control, with or without IgG addition (FIG. 8B). In this model, both CD3xCD20 bispecific antibodies show significant inhibition of tumor growth when IgG is added. As seen in FIG. 8A, the CD3xCD20-chimeric Fc bispecific antibody (Ab 1) demonstrates complete tumor growth inhibition during the study period with or without IgG supplementation in this experiment.

In FIG. 9 illustrates the regression of advanced tumors (˜200-400 mm ³ ) at day 14 in NSG mice treated with CD3xCD20 bispecific antibody. NSG mice were subcutaneously implanted with a mixture of Raji tumor cells and PBMC (HLA syngeneic cells) 15 days before treatment, then the tumors were allowed to develop and measured. Mice were treated with 0.4 mg/kg Antibody 1 (antibody CD3xCD20-chimeric Fc) or 0.4 mg/kg control Ab5 (anti-FeLD1 Ab), or stock control once a week (day 7, day 14, day 21) .

In FIG. 10 illustrates the regression of advanced tumors (~500-900 mm ³ ) at day 21 in NSG mice treated with CD3xCD20 bispecific antibody. NSG mice were subcutaneously implanted with a mixture of Raji tumor cells and PBMC (HLA syngeneic cells) 15 days before treatment, then the tumors were allowed to develop and measured. Mice were treated with 0.4 mg/kg Antibody 1 (antibody CD3xCD20-chimeric Fc) or 0.4 mg/kg control Ab5 (anti-FelD1 Ab), or stock control once a week (day 7, day 14, day 21) .

In FIG. 11A and 11B illustrate the in vivo efficacy of administration of the CD3xCD20 bispecific antibody, Antibody 1 and Antibody 4, compared with administration of a monospecific antibody (rituximab) by detecting changes in CD20+ B cell levels or CD3+ T cell levels in the peripheral blood of cynomolgus monkeys at day 7 research. Antibodies or placebo were administered on day 0. FIG. 11A: Peripheral blood B-cell levels were significantly reduced on day 2 in all samples except placebo; fig. 11B: On day 2, a transient decrease in the number of T cells was observed in the peripheral blood of animals treated with bispecific antibodies, which was restored to baseline levels on day 4. In the placebo or rituximab (Rituxan) treatment groups, no decrease in T-cell numbers (below baseline) was observed.

In FIG. 12A and 12B illustrate the in vivo efficacy of administering the CD3xCD20 bispecific antibody, Antibody 1 and Antibody 4, by detecting changes in CD20+ B cell levels or CD3+ T cell levels in peripheral blood of cynomolgus monkeys in a long-term (3 months) study. Placebo (stock solution) or bispecific antibodies were administered at 1.0 mg/kg on day 0. FIG. 12A: Peripheral blood B-cell levels were significantly reduced on day 2 and remained reduced throughout the study time in all samples except placebo; fig. 12B: For animals treated with bispecific antibodies, a temporary decrease in the number of T cells was observed on day 2, then on day 4, the number of T cells recovered to baseline levels and remained within baseline throughout the study (>80 days). Placebo-treated animals did not show a decrease in the number of T cells.

In FIG. 13A and 13B illustrate the in vivo efficacy of low dose administration of the CD3xCD20 bispecific antibody, Antibody 1 and Antibody 4, by detecting changes in CD20+ B cell levels or CD3+ T cell levels in peripheral blood of cynomolgus monkeys in a long-term (2 month) study. Bispecific antibodies were administered at 0.01 mg/kg or 0.001 mg/kg (1 μg/kg) on day 0. FIG. 13A: Peripheral blood B cell levels were significantly reduced on day 2 and remained reduced during the study time, similar to what was observed in animals treated with higher doses of CD3xCD20 bispecific antibodies (see also FIG. 11A or 12A); fig. 13B: Animals treated with very low doses (1 μg/kg) of the bispecific antibodies showed the same transient decrease in T cell numbers and recovery as observed in animals treated with higher doses of CD3xCD20 bispecific antibodies (also see Fig. 11B or 12B ).

In FIG. 14 illustrates the correlation of the decrease in the number of B cells with the decrease in the amount of antibody in the peripheral blood of animals treated with CD3xCD20-chimeric Fc Antibody 1. recover (eg, as observed at day 81 for animal #I06881 (solid circles)).

In FIG. 15 illustrates the correlation of the decrease in the number of B cells with the decrease in the amount of antibody in the peripheral blood of animals treated with 20-chimeric Fc Antibody 2. recover (eg, as observed at day 66 for animal #I06876 (solid triangles) and at day 68 for animal #I06877 (solid squares)).

In FIG. 16 illustrates the correlation of a decrease in the number of B cells with a decrease in the number of antibodies in the peripheral blood of animals treated with a CD3xCD20-gm Fc (Ab 4) bispecific antibody. As the amount of antibodies (open symbols) in the circulation of animals decreases over time, B cell populations (solid symbols) begin to recover (eg, as observed at day 91 for animal #I06870 (solid triangles) and at day 64 for animal #I06872 (filled squares)).

In FIG. 17A and 17B illustrate the reduction in tissue B cells in the spleen (FIG. 17A) or mesenteric lymph nodes (FIG. 17B) of cynomolgus monkeys following administration of CD3xCD20 bispecific antibodies compared to anti-CD20 monospecific antibody, at much lower doses (doses from 0.01 to 1.0 mg/kg) administered to bispecific groups. This reduction was not observed in any of the mentioned tissues of animals from the placebo-treated control group. Both

CD3xCD20 bispecific antibodies (Ab1 and Ab4) resulted in a dose-dependent decrease in the number of B cells in lymphoid organs, and doses of bispecific antibodies equal to or greater than 0.1 mg/kg reduced the number of B cells more effectively than rituximab.

In FIG. 18A and 18B illustrate that CD3xCD20 bispecific antibodies induce proliferation of human PBMC (Figure 18A) or cynomolgus monkey PBMC (Figure 18B) in an in vitro bioassay, while control Antibody 5 (-▲-; not specific for CD3xCD20) shows no activity. .

In FIG. 19A and 19B illustrate CD3xCD20-mediated targeted killing of Raji in a cytotoxicity assay. Antibody 1 mediated targeted cell killing with typical EC ₅₀ values of 25.0 pM and 9.10 pM for human (FIG. 19A) and cynomolgus monkey (FIG. 19B) T cells, respectively. Antibody 4 mediated targeted cell killing with typical EC ₅₀ values of 15.7 pM and 1.73 pM for human (FIG. 19A) and cynomolgus monkey (FIG. 19B) T cells, respectively. For the control (-▲ -), no activity was observed.

In FIG. 20A and 208B illustrate that the CD3xCD20 bispecific antibody mediates cell killing by naive T cells. In FIG. 20A illustrates a typical FACS scatter plot for the highest concentration of Antibody 4 tested. Wild-type B16F10.9 cells are labeled with CFDA-SE and B16F10.9/CD20 cells are labeled with Violet Cell Tracker. Effector cells do not label. In the second panel of Fig. 20A illustrates that CD20-expressing cells are killed (lower right quadrant) by treatment with anti-CD3xCD20. In FIG. 20B illustrates the proportion of surviving B16F10.9/CD20 cells after 48 hours in the presence of CD20xCD3 antibodies, ie. Antibody 4 (dt Fc), Antibody 1 (chimeric Fc) or control Ab 5, and PBMC. Percent survival was determined by comparing the percentage of B16F10.9/CD20 cells with CD20-negative B16F10.9 cells in a live cell population. Ab 4 and Ab 1 specifically directed human T cells to kill only CD20-expressing target cells (FIG. 20B) in a mixed cell population. Targeted cell killing was observed only in the presence of bispecific antibodies, with B16F10.9/CD20 cell numbers reduced in a dose-dependent manner by Antibody 4 (EC50 12.8 pM) and Antibody 1 (EC ₅₀ 19.5 pM) (FIG. 20B). Less than 5% of CD20-expressing cells remained alive at the highest dose studied (10 μg/ml).

In FIG. 21 illustrates the percentage of activated (CD69+) cells out of total CD2+ effector cells in a 48 hour cytotoxicity assay targeting B16F10.9/CD20 cells, with such activation being induced by any CD20xCD3 antibody, ie. Antibody 4 (dt Fc) or Antibody 1 (chimeric Fc).

In FIG. 22A and 22B illustrate that the CD3xCD20 bispecific antibody induced clustering of T cells with target cells (CD20+ cells) via bispecific binding arms. Effector cells are stained with CFSE and CD20+ cells are stained with Violet Cell Tracker and placed in separate quadrants. After incubation with an irrelevant control antibody (control Antibody 5), no clusters appeared in the cell mixture (double staining) (FIG. 22A). After incubation with the CD3xCD20 bispecific antibody (Ab 4), cell clusters appear as they are stained with both CFSE and Violet (see upper left quadrant of the scatter plot of FIG. 22B, highlighted by bold square).

In FIG. 23 illustrates a study of tumor volume (in mm ³ ) in NSG mice implanted subcutaneously with a mixture of Raji tumor cells and PBMCs, with the CD3xCD20 bispecific antibody of the invention (Ab 1) at 0.4 mg/kg, 2X/week (w.b .), an irrelevant control antibody Ab 6 at 0.4 mg/kg, 2X/week (ip) or stock solution was compared with rituximab, an anti-CD20 antibody at 8 mg/kg, 5X/week (ip) ) and CD19xCD3 BiTE at 0.5 mg/kg, 5X/week (i.v.). (For CD19xCD3 BiTE, see Nagorsen D, et al. Pharmacol Ther. 2012 Dec;136(3):334-42, 2012). Each was administered after tumor implantation. Data were expressed as mean (SPS) and ANOVA was performed. Ab1 dosed 2x per week ip was comparable to CD19xCD3 BiTE dosed 5k/week ip in this in vivo model.

In FIG. 24 illustrates a study of tumor volume (in mm ³ ) in NSG mice implanted subcutaneously with Raji/PBMC mixture, similar to FIG. 23, with ANOVA performed for AB1, control Ab 6, rituximab, and stock control solution. Ab 1, which was dosed 2x in

- 13 039429 week, was superior to rituximab therapy (dosed at 8 mg/kg; 5x/wk ip) in suppressing advanced Raji tumors.

In FIG. 25A and 25B illustrate tumor growth retardation when treatment was initiated simultaneously with or after hCD20/B16F10.9 tumor transplantation in humanized mice treated with CD3xCD20 bispecific antibody. Fig. 25A: hCD3 mice were implanted subcutaneously with hCD20-transduced B16F10.9 melanoma cells and simultaneously treated with 0.004 mg/kg or 0.4 mg/kg Antibody 1 (CD3xCD20 chimeric Fc antibody) or 4 mg/kg control Ab5 (anti-FeLD1 Ab) , or stock control solution (ip, 2 times a week). Fig. 25B: hCD3 mice were subcutaneously implanted with hCD20/B16F10.9 melanoma cells and developed tumors were treated on day 10 onwards with Antibody 1 (CD3xCD20 chimeric Fc antibody) or control. Mice twice a week i.b. treated with 0.4 or 4 mg/kg Ab1 or 0.4 mg/kg control Ab5 (anti-FelD1 Ab), or stock control solution.

Detailed description of the invention

Before the present invention is described, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as these methods and conditions may vary. It should also be understood that the terminology used herein is intended solely to describe specific embodiments of the invention and is not limiting, since the scope of the present invention is limited only by the appended claims.

Unless otherwise indicated, all technical and scientific terms used herein have the meanings normally implied by a person skilled in the art to which the invention belongs. In the context of this document, the term about, when applied to a particular given numerical value, means that the value may deviate from the given value by no more than 1%. For example, in the context of this document, an expression near 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

While any methods and materials similar or equivalent to those described herein may be used to practice or test the present invention, preferred methods and materials will now be described.

Definitions

In the context of this document, the expression CD3 refers to an antigen that is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed by association of two of the four receptor chains: CD3epsilon, CD3 delta, CD3 -zeta and CD3-gamma. All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the corresponding protein, polypeptide, or protein fragment, unless clearly stated to be of non-human origin. Thus, the expression CD3 means human CD3 unless it is stated to be of non-human origin, such as mouse CD3, simian CD3, etc.

As used herein, an antibody that binds to CD3 or an anti-CD3 antibody includes antibodies and antigen-binding fragments thereof that specifically recognize one of the CD3 units (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (eg, CD3 dimers gamma/epsilon, delta/epsilon, and zeta/zeta). The antibodies and antigen-binding fragments thereof according to the present invention can bind soluble CD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3 proteins as well as recombinant variants of CD3 proteins, such as, for example, monomeric and dimeric CD3 constructs that lack a transmembrane domain or are not otherwise associated with the cell membrane.

As used herein, cell surface expressed CD3 means one or more CD3 proteins that are expressed on the cell surface in vitro or in vivo such that at least a portion of the CD3 protein resides on the extracellular side of the cell membrane and is accessible to the antigen-binding portion of the antibody.

Cell-surface-expressed CD3 includes CD3 proteins that are part of a functional T-cell receptor in the cell membrane. Cell surface expressed CD3 expression includes CD3 protein expressed as part of a cell surface homodimer or heterodimer (eg, CD3 gamma/epsilon, delta/epsilon, and zeta/zeta dimers). Expression of cell surface-expressed CD3 also includes a CD3 chain (eg, CD3epsilon, CD3-delta, or CD3-gamma) that is expressed alone, without other types of CD3 chains, on the cell surface. Cell surface expressed CD3 may comprise or consist of a CD3 protein expressed on a cell surface that normally expresses the CD3 protein. Alternatively, cell-surface-expressed CD3 may comprise or consist of a CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface, but has been artificially engineered to express

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CD3 on its surface.

In the context of this document, the expression anti-CD3 antibody includes monovalent antibodies with single specificity, as well as bispecific antibodies containing a first arm that binds CD3 and a second arm that binds a second antigen (target), and the anti-CD3 arm contains any of sequences HCVR/LCVR or CDR, are given in this document in table. 1 or 2.

Examples of bispecific anti-CD3 antibodies are described elsewhere in this document. The term antigen-binding molecule includes antibodies and antigen-binding fragments of antibodies, including, for example, bispecific antibodies. Exemplary anti-CD3 antibodies are also described in US 2007/0280945A1; and PCT International Application No. PCT/US13/60511, filed September 19, 2013, which is hereby incorporated by reference in its entirety.

In the context of this document, the term CD20 refers to the human CD20 protein unless indicated to be of non-human origin (eg murine CD20, simian CD20, etc.). The human CD20 protein has an amino acid sequence corresponding to the standard NCBI sequence NP_690605.1.

In the context of this document, the expression anti-CD20 antibody includes monovalent antibodies with single specificity, such as Rituxan (rituximab), described in US patent 7879984. Exemplary anti-CD20 antibodies are also described in US patent 7879984 and PCT international application No. PCT/US13/ 60511, filed September 19, 2013, which are hereby incorporated by reference.

A tumor target antigen refers to a target antigen expressed by tumor cells, which may be expressed by a cognate cell (or healthy cells) prior to transformation into a tumor. A tumor-specific antigen refers to an antigen that is expressed or present in a tumor cell, but not normally present in a healthy cell or cells of other origins.

As used herein, the term antibody means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with a particular antigen (eg, CD3). The term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) and two light (L) chains linked to each other by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2 and C _H 3. Each light chain contains a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (C _L 1). The V _H and VL regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the FR of the anti-CD3 antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. The consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs.

In the context of this document, the term antibody also includes antigen-binding fragments of full-length antibody molecules. As used herein, the terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, etc. include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be obtained, for example, from full-length antibody molecules using any suitable standard methods, such as proteolytic cleavage or recombinant genetic engineering methods, including processing and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and/or readily available, for example from commercial sources, DNA libraries (including, for example, antibody phage libraries), or can be synthesized. The DNA can be sequenced and processed chemically or by molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc.

Non-limiting examples of antigen-binding fragments include:

(i) Fab fragments;

(ii) F(ab')2 fragments;

(iii) Fd fragments;

(iv) Fv fragments;

- 15 039429 (v) single chain Fv molecules (scFv);

(vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., a distinguished complementarity determining region (CDR) such as a CDR3 peptide), or a conformationally constrained FR3-CDR3-FR4 peptide freedom. Other engineered molecules such as domain specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.) , immunopharmaceuticals based on small modular proteins (SMIP) and shark IgNAR variable domains are also included in the expression antigen-binding fragment in the context of this document.

An antigen binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or amino acid composition, and generally contains at least one CDR that is contiguous or in-frame with one or more framework sequences. In antigen-binding fragments in which a VH domain is linked to a V _L domain, the VH and V _L domains may be in any suitable location relative to each other. For example, the variable region may be dimeric and contain V _H -V _H , V _H -V _L or V _L -V _L dimers. Alternatively, the antigen-binding fragment of an antibody may comprise a V _H or V _L monomeric domain.

In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant fragment (Fc) domain or otherwise linked to the Fc domain. Non-limiting exemplary variable and constant domain configurations that can be found within an antigen-binding fragment of an antibody of the present invention include: (i) V _{H -CH} 1-hinge region- _CH 2 _-CH 3; (ii) VH-hinge region-C _H 2-C _H 3; (iii) V _H -C _L ; (iv) V _L -C _H 1CH2-CH3; (v) VL-CH2-CH3; and (vi) V _L -C _L . In any configuration of the variable and constant domains, including any of the above exemplary configurations, the variable and constant domains can either be directly linked to each other, or can be connected (linked) by a complete or partial chimeric hinge region according to the invention, or a complete or partial CH1- region and chimeric hinge region. The hinge region may be composed of at least upper and lower hinge amino acids, resulting in a flexible or semi-flexible connection between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations above in non-covalent association with each other and/or with one or more VH or VL monomeric domains (e.g. , via disulfide bonds).

The multispecific antibody format of the invention, including the exemplary bispecific antibody formats disclosed herein, typically contains at least two different variable domains, with each variable domain being able to specifically bind to a different antigen. Multispecific formats can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using conventional techniques known in the art.

The antibodies of the present invention are modified to have reduced or no complement dependent cytotoxicity (CCC) or antibody dependent cell mediated cytotoxicity (ADCC) as measured by in vitro measurements. Complement dependent cytotoxicity (CDC) refers to the lysis of antigen expressing cells by an antibody of the invention in the presence of complement. Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated response in which non-specific cytotoxic cells that express Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on the target cell and thereby leading to lysis of the target cell. The measurement of CSC and ADCC can be carried out using methods of analysis that are well known and available in the art. (See, for example, US Pat. Nos. 5,500,362 and 5,821,337 and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95: 652-656). The heavy chain constant region (CH) of an antibody is important for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Therefore, the CH antibody can be selected based on the need for the antibody to mediate cytotoxicity.

In certain embodiments of the invention, the bispecific antibodies of the invention are human antibodies. As used herein, the term human antibody includes antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation), for example, in CDRs and in particular CDR3. However, in the context of this document, the term human antibody does not include antibodies in which CDR sequences derived from the germline of other mammalian species, such as mice, have been grafted into human framework sequences.

Antibodies of the invention may, in some embodiments, be recombinant human antibodies. For the purposes of this document, the term recombinant human antibody is intended to include all human antibodies that are produced, expressed, generated or isolated by recombinant methods, such as antibodies expressed with a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res 20: 6287-6295), or antibodies that are made, expressed, made or isolated by any other method that involves splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments of the invention, recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of an animal transgenic for human Ig sequences, in vivo somatic mutagenesis) and. thus, the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences which, although derived from and related to human germline _VH and VL sequences, may not exist naturally in the human germline antibody repertoire in vivo.

Human antibodies can exist in two forms, which are associated with the heterogeneity of the hinge region. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa, in which the dimers are held together by heavy chain inter-chain disulfide bonds. In the second form, the dimers are not connected by interchain disulfide bonds, and the molecule of about 75-80 kDa consists of covalently coupled light and heavy chains (semi-antibody). These forms are very difficult to separate even after affine purification.

The frequency of manifestation of the second form in different isotypes of intact IgG is associated, without limitation, with structural differences associated with the isotype of the hinge region of the antibody. A single amino acid substitution in the human IgG4 hinge region can significantly reduce expression of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels normally seen with the human IgG1 hinge region. The present invention includes antibodies containing one or more mutations in the hinge region, which can, for example, when produced, improve the yield of the desired form of the antibody.

The antibodies of the invention may be isolated antibodies. As used herein, an isolated antibody means an antibody that has been identified and separated and/or separated from at least one component of its natural environment. For example, an antibody that has been separated or separated from at least one component of an organism, or from a tissue or cell in which the antibody is normally present or naturally produced, is an "isolated antibody" for the purposes of the present invention. An isolated antibody also includes an antibody in situ in a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In accordance with certain embodiments of the invention, the isolated antibody may be substantially free of other cellular material and/or chemicals.

Ahtu-CD3 or anti-CD20 variable regions disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences, from which antibodies were obtained. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof, which are derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding germline sequence residue(s). from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (in this document, such changes in sequences are collectively referred to as mutations germ line). Starting from the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can easily generate numerous antibodies and antigen binding fragments that contain one or more individual germline mutations, or combinations thereof. In certain embodiments, all framework and/or CDR residues within the VH and/or V _L domains are mutated back to residues present in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to residues of the original germline sequence, for example, only mutated residues within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues within CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated to corresponding residues of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the invention may contain any combination of two or more germline mutations within the framework regions and/or CDR regions, for example, where some individual residues are mutated to the corresponding residue of a particular germline sequence, while some other residues that different from the original germline sequence, retained or mutated to the corresponding residue of a different germline sequence. Once generated, antibodies or antigen-binding fragments that contain one or more germline mutations can readily be tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate). ), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by such a general method are included in the present invention.

The present invention also includes anti-CD3 or anti-CD20 variable regions containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes anti-CD3 antibodies having the amino acid sequences of HCVR, LCVR, and/or CDR, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions compared to the amino acid sequences of HCVR, LCVR and/or CDR shown in table. one.

The term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. One antigen may have more than one epitope. Thus, different antibodies may bind to different sites on the antigen and may have different biological effects. Epitopes may be conformational or linear. A conformational epitope is formed by spatially combined amino acids from different segments of a linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. Under certain conditions, an epitope on an antigen may contain saccharide components, phosphoryl groups, or sulfonyl groups.

The term significant identity or substantially identical, when applied to a nucleic acid or fragment thereof, indicates that, when optimally aligned with the corresponding nucleotide insertions or deletions with another nucleic acid (or its complementary strand), the nucleotide sequence identity is at least 95% and more preferably at least 96%, 97%, 98%, or 99% nucleotide bases as determined by any well known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule that has a significant degree of identity with a standard nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the standard nucleic acid molecule.

When applied to polypeptides, the term significant similarity or substantially similar means that two peptide sequences, when optimally aligned, such as provided by the GAP or BESTFIT programs using default gap penalties, have at least 95% sequence identity, and even more. preferably at least 98 or 99% sequence identity. Preferably, the positions of the non-identical residues are distinguished by conservative amino acid substitutions. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R-group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution does not lead to significant changes in the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to account for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art.

- 18 039429 of the technical field. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine and tryptophan; (5) main side chains: lysine, arginine and histidine; (6) acid side chains: aspartate and glutamate, and (7) sulfur side chains have cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, also referred to as sequence identity, is typically determined using sequence analysis software. Protein analysis software contrasts similar sequences using similarity scores assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as Gap and Bestfit that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignment and percent sequence identity for regions with the best overlap between query and search sequences (Pearson (2000), supra). Another preferred algorithm for comparing a sequence according to the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN with default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

Bispecific antigen-binding molecules

The antibodies of the invention may be bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of the same target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The Ahtu-CD3xCD20 antibodies of the present invention may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or antibody fragment may be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to form a bispecific or multispecific antibodies with additional binding specificity.

Thus, the present invention includes bispecific antibodies in which one arm of the immunoglobulin binds human CD3 and the other arm of the immunoglobulin is specific for the target antigen. The target antigen that binds the other arm of the anti-CD3 bispecific antibody can be any antigen expressed on or near the cell, tissue, organ, microorganism, or virus against which a targeted immune response is desired. The CD3 binding arm may contain any of the HCVR/LCVR or CDR amino acid sequences shown in Table 1. one.

In the context of the bispecific antibodies of the present invention, in which one arm of the antibody binds CD3 and the other arm binds the target antigen, the target antigen may be a tumor-associated antigen. Non-limiting examples of tumor associated antigens include, for example, AFP, ALK, BAGE proteins, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20(MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, proteins GAGE (eg, GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins (eg, MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Mucl, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125 ), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), proteins RAGE, Ras, RGS5, Rho, SART-1, SART-3,

- 19 039429

Steap-1, Steap-2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

In the context of the bispecific antibodies of the present invention, in which one arm of the antibody binds CD3 and the other arm binds the target antigen, the target antigen may be an infectious disease-associated antigen. Non-limiting examples of infectious disease-associated antigens include, for example, an antigen that is expressed on the surface of a viral particle, or preferably expressed on a cell infected with a virus, the virus being selected from the group consisting of HIV, hepatitis (A, B or C), herpes virus (e.g., HSV-1, HSV-2, CMV, HAV-6, B3B, Espshein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, Coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T cell leukemia virus (HTLV), dengue virus, human papillomavirus, mollusk virus, poliovirus, rabies virus, John Cunningham virus and arbovirus encephalitis virus. Alternatively, the target antigen may be an antigen that is expressed on the surface of a bacterium or is preferably expressed on a cell infected with the bacterium, wherein the bacterium is selected from the group consisting of chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonella, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospira and the bacteria that cause Lyme disease. In certain embodiments, the target antigen is an antigen that is expressed on the surface of a fungus, or is preferably expressed on a fungal-infected cell, the fungus being selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.) , Crytococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (mucor, absidia, rhizopus, etc.), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum. In certain embodiments of the invention is an antigen that is expressed on the surface of the parasite, or preferably expressed on a cell infected with the parasite, while the parasite is selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp. , Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis, Taenia crassiceps and Brugia malayi.

Non-limiting examples of specific pathogen-associated antigens include, for example, HIV gp120, HIV CD4, hepatitis B glucoprotein L, hepatitis B glucoprotein M, hepatitis B glucoprotein S, hepatitis C E1, hepatitis C E2, hepatocyte-specific protein, herpes simplex virus gB, cytomegalovirus gB and HTLV envelope protein.

In accordance with certain exemplary embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind CD3 and CD20. Such molecules may be referred to herein, for example, as anti-CD3/anti-CD20 or anti-CD3/CD20 or anti-CD3xCD20 or CD3xCD20 bispecific molecules, or other similar terms.

As used herein, the expression antigen-binding molecule means a protein, polypeptide, or molecular complex containing or consisting of at least one complementarity-determining region (CDR) that alone or in combination with one or more additional CDRs and/or framework regions (FRs) specifically binds to a specific antigen. In certain embodiments of the invention, the antigen-binding molecule is an antibody or antibody fragment in the sense in which these terms have been defined elsewhere in this document.

In the context of this document, the expression bispecific antigennegative molecule means a protein, polypeptide or molecular complex containing at least a first antigennegative domain and a second antigennegative domain. Each antigen-binding domain in the bispecific antigen-binding molecule contains at least one CDR that, alone or in combination with one or more additional CDRs and/or FRs, specifically binds to a particular antigen. In the context of the present invention, a first antigen-binding domain specifically binds a first antigen (eg, CD3) and a second antigen-binding domain specifically binds a second, distinct antigen (eg, CD20).

In certain exemplary embodiments of the present invention, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody contains a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule comprising a first and a second antigen-binding domain (eg, a bispecific antibody), the CDRs of the first antigen-binding domain may be denoted by the prefix A1 and the CDRs of the second antigen-binding domain may be denoted by the prefix A2. Thus, the CDRs of the first antigen-binding domain may be referred to as A1-HCDR1, A1-HCDR2 and A1-HCDR3; a CDRs of the second antigen-binding domain may be referred to as A2-HCDR1, A2-HCDR2 and A2-HCDR3.

The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked to each other and form a bispecific antigen-binding molecule of the present invention (ie, a bispecific ScFv) further associated with an Fc domain. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be associated with a separate Fc domain. The bispecific antigen-binding molecules of the present invention typically contain two Fc domains, each of which is an individual part of a separate antibody heavy chain. The first and second Fc domains may have the same sequence, except for the presence of a mutation in the _CH 3 domain designed to simplify or facilitate the purification of heterodimeric (ie, bispecific) molecules.

The present invention includes bispecific antigen-binding molecules comprising a first C _H 3 domain and a second Ig C _H 3 domain, wherein the first and second Ig C _H 3 domains differ from each other by at least one amino acid and at least one amino acid the difference reduces the binding of the bispecific antibody to protein A compared to the bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes protein A binding, such as an _H435R modification (according to EU numbering; H95R according to IMGT exon numbering). The second CH3 may additionally contain the modification Y436F (according to EU numbering; Y96F according to IMGT). Additional modifications that may be present in the second Ch3 include: D356E, L358M, N384S, K392N, V397M and V422I according to EU (D16E, L18M, N44S, K52N, V57M and V82I according to IMGT) in the case of IgG1 CH3 domains; and Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU (Q15R, N44S, K52N, V57M, R69K, E79Q and V82I according to IMGT) in the case of IgG4 C _H 3 domains.

Other formats or technologies of bispecific antibodies can be used to produce antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or some other manner) to one or more other molecular moieties, such as another antibody or antibody fragment. , characterized by the binding specificity of the second antigen, to obtain a bispecific antigen-binding molecule. Particular exemplary bispecific formats that can be used in the context of the present invention include, without limitation, for example, scFv or diabodies based bispecific formats, IgG-scFv fusion products, Ig double variable domain (DVD), quadroma, ridge-in-trough , common light chain (e.g., common light chain with tab-in-gap, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, duobody, IgG1/IgG2, Fab (DAF)-IgG dual action and bispecific Mab ² formats (see, for example, Klein et al. 2012, mAb 4:6, 1-11 and references therein for an overview of the above formats).

In the context of the bispecific antigen-binding molecules of the present invention, Fc domains may contain one or more amino acid changes (eg, insertions, deletions, or substitutions) from a particular chimeric version of the Fc domain without changing the desired functionality. For example, the invention includes bispecific antigen-binding molecules containing one or more modifications in the Fc domain, which results in a modified Fc domain with a modified binding interaction (eg, increased or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen binding molecule comprises a modification in the CH2 or CH3 region, wherein the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in the endosome where the pH is in the range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (eg L/Y/F/W or T), 254 (eg S or T) and 256 (eg S/R/Q/E/D or T); or a modification at position 428 and/or 433 (eg L/R/S/P/Q or K) and/or 434 (eg H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (eg, 308F, V308F) and 434. In one embodiment, the modification includes modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); and modification 307 and/or 308 (eg, 308F or 308P).

Sequence Options

The antibodies and bespecifically antigen-binding molecules of the present invention may contain one or more amino acid substitutions, insertions and/or deletions in the framework regions and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the individual antigen-binding molecules were derived.

- 21 039429 domains. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The antigen-binding molecules of the present invention may comprise antigen-binding domains that are derived from any exemplary amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding residue(s) of the sequence. the germline from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to as germline mutations). lines). Starting with the heavy and light chain variable region sequences disclosed herein, one of ordinary skill in the art can readily generate numerous antibodies and antigen binding fragments that contain one or more individual germline mutations, or combinations thereof. In certain embodiments, all framework and/or CDR residues within the VH and/or V _L domains are mutated back to residues present in the original germline sequence from which the antigen binding domain was derived. In other embodiments, only certain residues are mutated back to residues of the original germline sequence, for example, only mutated residues within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only mutated residues within CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated to corresponding residues of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antigen-binding domain was originally derived). In addition, antigen-binding domains may contain any combination of two or more germline mutations within the framework regions and/or CDR regions, for example, where some individual residues are mutated to the corresponding residue of a particular germline sequence, while some other residues that differ from the original germline sequence, retained or mutated to the corresponding residue of another germline sequence. Once generated, antigen-binding domains that contain one or more germline mutations can readily be tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate), reduced immunogenicity, etc. Bispecific antigen-binding molecules containing one or more antigen-binding domains obtained in such a general way are included in the present invention.

The present invention also includes antigen-binding molecules in which one or both antigen-binding domains contain variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present invention includes antigen-binding molecules comprising an antigen-binding domain having the amino acid sequences HCVR, LCVR, and/or CDR, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions compared to the HCVR, LCVR and/or CDR amino acid sequences disclosed herein. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R-group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution does not lead to significant changes in the functional properties of the protein. Examples of groups of amino acids that have side chains with similar chemical properties include:

(1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine;

(2) aliphatic hydroxyl side chains: serine and threonine;

(3) amide-containing side chains: asparagine and glutamine;

(4) aromatic side chains: phenylalanine, tyrosine and tryptophan;

(5) main side chains: lysine, arginine and histidine;

(6) acid side chains: aspartate and glutamate and (7) sulfur side chains have cysteine and methionine. Preferred groups of conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysinarginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, also referred to as sequence identity, can be determined using sequence analysis software. Protein analysis software contrasts similar sequences using similarity scores assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as Gap and Bestfit that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA using default or recommended parameters; program in GCG version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignment and percent sequence identity for regions with the best overlap between query and search sequences (Pearson (2000), supra). Another preferred algorithm for comparing a sequence according to the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN with default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.

pH dependent binding

The present invention includes anti-CD3/anti-CD20 bispecific antibodies with pH dependent binding characteristics. For example, an anti-CD3 antibody of the present invention may exhibit reduced binding to CD3 at acidic pH compared to neutral pH. Alternatively, anti-CD3 antibodies of the invention may exhibit increased binding to CD3 at acidic pH compared to neutral pH. The expression acidic pH includes pH values less than about 6.2, for example, about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5, 0 or less. As used herein, neutral pH means a pH of about 7.0 to about 7.4. The expression neutral pH includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35 and 7.4.

In certain cases, reduced binding ... at acidic pH compared to neutral pH is expressed in terms of the ratio of the KD value of an antibody's binding to its antigen at acidic pH to the KD value of an antibody's binding to its antigen at neutral pH (or vice versa). For example, an antibody or antigen-binding fragment thereof may be considered to exhibit reduced binding to CD3 at acidic pH compared to neutral pH for the purposes of the present invention if the antibody or antigen-binding fragment thereof exhibits an acid/neutral Kd ratio of 3.0 or greater. In certain exemplary embodiments, the acid/neutral Kd ratio for an antibody or antigen-binding fragment of the present invention may be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12, 5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

Antibodies with pH-dependent binding characteristics can be obtained, for example, by screening an antibody population for reduced (or increased) binding to a particular antigen at acidic pH compared to neutral pH. In addition, modifications to the antigen-binding domain at the amino acid level can result in antibodies with pH-dependent binding characteristics. For example, an antibody with reduced antigen binding at acidic pH compared to neutral pH can be obtained by replacing one or more amino acids of the antigen binding domain (eg, in the CDR) with a histidine residue.

Fc receptor binding

Anti-CD3/anti-CD20 bispecific antigen-binding molecules and antibodies of the invention are provided, containing a chimeric Fc domain such as a hinge region-CH2-CH3 Ig heavy chain constant domain derived from various IgG isotypes and having unique binding and activation characteristics. Fc receptor. Certain Fc domains according to the invention are designed to contain a chimeric hinge region.

In the context of this document, the term chimeric means consisting of parts of different origin. The expression chimeric protein includes a first amino acid protein associated with a second amino acid protein with which it is not normally associated in nature. The amino acid sequences can typically exist as separate proteins or in a different order within the same protein and be joined in a new order in the fusion polypeptide. Chimeric proteins can be created by various methods known in the art, for example, by chemical synthesis or by creating a polynucleotide that encodes the amino acids of the chimeric protein in the desired order. Exemplary chimeric proteins include chimeric hinge sequences connecting IgG heavy chain domains and fusion proteins engineered to produce human antibodies and antigen binding proteins of the present invention.

The chimeric proteins disclosed herein were designed to minimize the formation of immunogenic epitopes in compounds, for example, compared to the Fc region or

- 23 039429 wild-type IgG domain. Therefore, engineered proteins according to the invention have reduced immunogenicity and exhibit reduced binding to Fc receptors as well as reduced or absent effector functions.

As used herein, the term hinge region is meant to include the region of consecutive amino acid residues that connect the C-terminus of the CH1 domain to the N-terminus of the CH2 domain of an immunoglobulin. Several amino acids at the N-terminus of the CH2 domain, which are encoded by the CH2 exon, are also considered to be part of the lower hinge region. Without wishing to be bound by any one theory, the hinge amino acids IgG1, IgG2, and IgG4 have been characterized as containing 12-15 consecutive amino acids encoded by a single hinge exon and several N-terminal amino acids of the CH2 domain (encoded by the CH2 exon) (Brekke, O.H., et al. Immunology Today 16(2):85-90 (1995)). On the other hand, IgG3 contains a hinge region consisting of four segments: one upper segment, resembling the hinge region of IgG1, and 3 segments identical to the amino acid repeats characteristic of IgG3.

As used herein, the term chimeric hinge is intended to include a chimeric protein comprising a first amino acid sequence derived from the hinge region of one Ig molecule and a second amino acid sequence derived from the hinge region of an Ig molecule of another class or subclass. Exemplary chimeric hinges of the present invention comprise a first amino acid or upper hinge sequence derived from a human IgG1 hinge or human IgG4 hinge and a second lower hinge amino acid or sequence derived from a human IgG2 hinge. In certain embodiments of the invention, the first or upper hinge sequence contains amino acid residues at positions 216 to 227, according to EU numbering. In some embodiments, the second or lower hinge sequence contains amino acid residues at positions 228 to 236, according to EU numbering.

For the purposes of this invention, the upper hinge region is meant to contain amino acid residues at positions 216 to 227 according to EU numbering (amino acid residues at positions 226 to 240 according to Kabat numbering) (see FIG. 1). The lower hinge region is meant to contain amino acid residues at positions 228 to 236 according to EU numbering (amino acid residues at positions 241 to 249 according to Kabat numbering) (see FIG. 1).

In this document, for the convenience of the person practicing the invention, the amino acid residues of the hinge region for human IgG1, IgG2 and IgG4 have been defined according to Kabat's EU numbering system (Kabat, EA et al., Sequences of Proteins of Immunological interest. 5 ^th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991)), also known as EU numbering or EU index, updated according to IMGT® Scientific Chart, IMGT®, International Immunogenetic Information System®, http: //www. imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.ht ml Created: May 17, 2001 Last updated: January 10, 2013

The correspondence between EU numbering for human IgG1, IgG2, and IgG4 hinge amino acids and the IMGT unique domain numbering, IMGT exon numbering, and Kabat numbering systems (see also Kabat, E.A. et al., 1991, supra) are described in Table 1. A-F below:

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Table A. IgGl hinge region numbering

Amino acids IgGl (IGHG1) [SwissProt Р01857] Unique numbering FOR HINGE AREA IMGT ^a Exon numbering IMGT ^a EU numbering Kabat numbering (E) one one 216 226 R 2 2 217 227 To 3 3 218 228 S four four 219 232 ^a [ 229] ^b FROM 5 5 220 233 ^a [ 230] ^b D 6 6 221 234 ^a [232] ^b To 7 7 222 235 T eight eight 223 236 n 9 9 224 237 t ten ten 225 238 With eleven eleven 226 239 R 12 12 227 240 R 13 13 228 241 With fourteen fourteen 229 242 R fifteen fifteen 230 243

Table B. IgGl C-domain hinge region numbering

Amino acids IgGl (IGHG1) [SwissProt P01857] Unique numbering for C-domains IMGT ^a Exon numbering IMGT ^a EU numbering Numbering Kabata (BUT) 1.6 one 231 244 R 1.5 2 232 245 E 1.4 3 233 246 L 1.3 four 234 247 L 1.2 5 235 248 G 1.1 6 236 249

Table C. Numbering of the IgG2 hinge region

Amino acids IgG2 (IGHG2) Unique numbering Numbering Numbering Numbering [SwissProt P01859] FOR HINGE AREA IMGT ^a i exon imgt ^a EU Kabata (E) one one 216 226 R 2 2 217 227 To 3 3 218 228 FROM four four 219 ^a (221) ^b 232 FROM 5 5 220 ^a (-) ^b 233 V 6 6 222 235 E 7 7 224 237 FROM eight eight 226 239 R 9 9 227 240 R ten ten 228 241 FROM eleven eleven 229 242 R 12 12 230 243

Table D. Numbering of the IgG2 C-domain hinge region

Amino acids IgG2 (IGHG2) [SwissProt P01859] EU numbering Kabat numbering (BUT) 231 244 R 232 245 R 233 246 V 234 247 BUT 235 248 — 236 249

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Table E. IgG4 hinge region numbering

Amino acids IgG4 (IGHG4) [SwissProt P01861] Unique numbering for HINGE AREA IMGT ^a IMGT exon numbering ^a EU numbering Kabat numbering (Ε) one one 216 226 S 2 2 217 227 To 3 3 218 228 Y four four (219) ^b 229 G 5 5 (220) ^b 230 P 6 6 224 237 P 7 7 225 238 C eight eight 226 239 P 9 9 227 240 S ten ten 228 241 C eleven eleven 229 242 P 12 12 230 243

Table F. IgG4 C-domain hinge region numbering

Amino acids IgG4 (IGHG4) [SwissProt P018 61] EU numbering Kabat numbering (BUT) 231 244 R 232 245 E 233 246 F 234 247 L 235 248 G 236 249

Amino acids obtained by exon splicing are indicated in brackets.

- means no corresponding number

-- indicates the absence of the corresponding amino acid at that position ^a numbering according to the latest update of the IMGT Scientific Chart ^b numbering according to the EU index given in Kabat, EA, et al. 1991

See also, for example, Lefranc, M.-P. et al., Devel Comp Immunol, 29, 185-203 (2005); and Edelman, G.M. et al. PNAS USA, 63:78-85 (1969).

The term binding, in the context of binding an antibody, Ig, antibody-binding fragment, or Fc-containing protein, e.g., to a given antigen or FcyR, generally refers to an interaction or association between at least two components or molecular structures, such as an antibody-antigen interaction or Fc-containing protein and FcyR.

For example, the binding affinity typically corresponds to a Kd value of about 10 ^-7 M or less, for example, about 10 ^-8 M or less, for example, about 10 ^-9 M or less, as determined by the method, for example, surface plasmon resonance (SPR) on a BIAcore 3000 instrument using an antigen or FcR as ligand and an antibody, Ig binding antibody fragment, or Fc containing protein as analyte (or anti-ligand). Accordingly, an antibody or other binding protein binds to a given antigen or receptor with an affinity corresponding to a Kd value that is at least ten times lower, such as at least 100 times lower, such as at least 1000 times lower, such as , at least 10,000 times lower, for example, at least 100,000 times lower than its binding affinity for a non-specific antigen (eg, BSA, casein).

In the context of this document, the term Kd (M) refers to the equilibrium dissociation constant of a particular antibody-antigen interaction or the equilibrium dissociation constant of an antibody, Ig, antibody binding fragment, or Fc-containing protein with FcyR. There is an inverse relationship between Kd and binding affinity, therefore, the lower the Kd value, the higher (ie, stronger) the affinity. Thus, the terms higher affinity or stronger affinity refer to a higher ability to form an interaction and hence a lower Kd, and conversely, the terms lower affinity or weaker affinity refer to a lower ability to form an interaction and hence a higher Kd. . In some cases, a higher binding affinity (or Kd) of a particular molecule (for example, an antibody) for its associated partner molecule (for example, the X receptor) compared to the binding affinity of a molecule (for example, an antibody) for another associated partner molecule (for example, , receptor Y) can be expressed as a ratio of binding, determined by dividing the larger Kd value (lower or weaker affinity) by the smaller Kd value (higher or stronger affinity), for example, expressing as 5-fold or 10-fold higher affinity depending on the circumstances.

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In the context of this document, the term k _d (c-1 or 1/c) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody, Ig, antibody binding fragment, or Fc-containing protein with FcyR. This value is also called the value k _du cc.

As used herein, the term ka (M-1 x _c -1 or 1/M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody, Ig, antibody binding fragment, or Fc-containing protein to FcyR.

As used herein, the term kA (M-1 or 1/M) refers to the equilibrium association constant of a particular antibody-antigen interaction, or the equilibrium association constant of an antibody, Ig, antibody binding fragment, or Fc-containing protein to FcyR. The equilibrium association constant is obtained by dividing k _a by k _d .

In the context of this document, the term EC ₅₀ or EC ₅₀ refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a response midway between baseline and maximum after a certain exposure time. EC ₅₀ generally represents the concentration of an antibody at which 50% of its maximum effect is observed. Thus, reduced binding is observed at elevated EC ₅₀ or half maximum effective concentration.

In one embodiment of the invention, reduced binding can be defined as an increased concentration of EC ₅₀ antibodies, which provides binding to half the maximum number of target cells.

In some embodiments of the invention, reduced cytotoxic activity, such as ADCC or CDC, can be defined as an increased concentration of EC ₅₀ antibodies, which leads to the lysis of half the maximum number of target cells. Cytotoxicity is also defined as percent cytotoxicity or percent lysis, which is the proportion of the total cell population defined as lysed in a calcein release assay or equivalent assay. The percentage of cytotoxicity can be determined as described in example 6.

In other embodiments of the invention, reduced proliferation can be defined as an increased concentration of EC50 antibodies, which allows the proliferation of half the maximum number of target cells.

In the context of this document, the expression effector functions is meant to include the functionalities provided by the Fc-containing protein after FcyR binding. Without wishing to be bound by any one theory, the formation of the Fc/FcyR complex recruits a large number of effector cells at antigen bound sites, which typically results in a variety of intracellular signaling events and important subsequent immune responses.

The chimeric Fc-containing antigen-binding proteins and antibodies of the invention exhibit altered or reduced effector functions compared to the corresponding wild-type Fc-containing antigen-binding proteins or antibodies. See, for example, PCT Publication No. WO 2014/121087, published August 7, 2014, which is incorporated herein by reference in its entirety.

In some embodiments, the altered or reduced effector function is a cytotoxic effector function, such as cytotoxicity, complement-dependent cytotoxicity (CCC), or antibody-dependent cytotoxicity (ADCC). In one embodiment of the invention, the altered or reduced effector function is complement dependent cytotoxicity. In another embodiment of the invention, the altered or reduced effector function is antibody-dependent cytotoxicity. In other embodiments of the invention, the altered or reduced effector function is cellular proliferation of target cells.

Several antibody effector functions are mediated, at least in part, by Fc receptors (FcRs), which bind the Fc region of an antibody in the constant domain (particularly the CH2 and CH3 domain) of a typical immunoglobulin. There are a large number of Fc receptors that are specific for different classes of immunoglobulins, i.e. IgG, IgE, IgA, IgM and IgD. The human IgG F receptor family is divided into three groups: FcyRI (CD64), which is able to bind IgG with high affinity, FcyRII (CD32) and FcyRIII (CD16), which are both low affinity receptors. Each FcyR subclass is encoded by two or three genes, and alternative RNA splicing results in multiple transcripts, and hence there is a wide variety of FcyR isoforms (e.g., FcyRIA (CD64; FCGR1A), FcyRIB (CD64; FCRG1B), FcyRIIA (CD32A; FCGR2A ), FcyRIIB (CD32B; FCGR2B), FcyRIIC (CD32C; FCGR2C), FcyRIIIA (CD16a; FCGR3A) and FcyRIIIB (CD16b; FCGR3B)). In addition, FcRn or neonatal Fc receptor (also known as Fc receptor transporter, alpha, or FCGRT) is able to transfer IgG antibodies from the mother to the fetus across the placenta. In addition, Fc receptors are expressed on a wide variety of cells, including, for example, B cells, monocytes, dendritic cells, neutrophils, and some lymphocytes. For example, U937 cells of the human monocyte cell line express both FcyRI and FcyRIIA (see, for example, Jones, et al. J Immunol 135(5):3348-53 (1985); and Brooks, et al. J Exp Med 170:1369-85 (October 1989)). Each receptor referred to herein includes any known functional form of the receptor, including transcript variants, isoforms, and polymorphisms.

Binding of Fc Ig to its receptor leads these effector cells to antigen binding sites, which ultimately leads to a variety of signaling and immune responses, including B-cell activation, inflammatory responses, cytotoxic responses, and phagocytic responses. Therefore, reduced or altered Fc Ig binding to its receptor may lead to reduced effector functions.

The term antibody-dependent cellular phagocytosis or ADCP refers to an effector function that destroys (or kills) a target cell by engulfing the target cell rather than inducing cytolysis. ADCP may be an important in vivo mechanism for the destruction of tumor cells. ADCP can be determined by two-color fluorescent flow cytometry methods, for example, methods using, for example, PKH2 (green fluorescent dye) and phycoerythrin (red) conjugated monoclonal antibodies against various cell surface proteins to separate the studied cells, thus determining phagocytic activity and the rate of phagocytosis. ADCF measurements are well known in the art. Therapeutic strategies that selectively activate FcyRIIa over FcyRIIb can enhance the phagocytic activity of macrophages (Richards, JO, et al. 2008 Mol Cancer Ther 7(8):2517-27). The chimeric Fc-containing antigen-binding proteins and antibodies of the invention bind and activate human FcyRIIA. In certain instances, the antigen-binding proteins and antibodies of the invention bind human FcyRIIA with greater affinity than these antibodies bind FcyRIIB. In some embodiments, the antibody exhibits a Kd binding affinity for human FcyRIIA greater than 5-fold greater than the KD binding affinity for human FcyRIIB. In other embodiments, the antibody exhibits a Kd binding affinity for human FcyRIIA greater than about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times Kd binding affinity for human FcyRIIB.

Biological characteristics of antibodies and bispecific antigen-binding molecules

The present invention includes antibodies and antigen-binding fragments thereof that bind human CD3 and CD20. For example, the present invention includes anti-CD3xCD20 antibodies that bind Jurkat (CD3+) cells and Raji (CD20+) cells with an EC ₅₀ value of less than about 60 nM as measured by an in vitro binding assay, e.g., the assay format described in Example 3 of this document (eg, evaluation of binding of Jurkat cells or Raji cells to CD3xCD20 antibodies), or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind CD3 or CD20 on the surface of a cell (e.g., Jurkat cells and/or Raji cells) with an EC ₅₀ value of less than about 75 nM, less than about 70 nM, less than about 65 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, or less than about 25 nM, as determined by an in vitro binding assay, e.g., the assay format described in Example 4 of this document, or a substantially similar analysis.

The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies that are capable of simultaneously binding to human CD3 and human CD20. In accordance with certain embodiments of the invention, the bispecific antigen-binding molecules of the invention interact specifically with cells that express CD3 and/or CD20. in which the bispecific antigen binding molecule binds cells that express CD3 and/or CD20 can be assessed using fluorescence activated cell sorting (FACS) as illustrated in Example 4 of this document.For example, the present invention includes bispecific antigen binding molecules that specifically bind human T cell lines that express CD3 (eg, Jurkat), human B cell lines that express CD20 (eg, Raji), and primate T cells (eg, peripheral blood mononuclear cells [MCPC] cynomolgus macaque).

The present invention includes bispecific antigen binding molecules that bind any of the above cells and cell lines with an EC ₅₀ value of from about 8.74 x 10' ⁶ to about 5.99 x 10' ⁸ or less according to the FACS analysis described in Example 4, or in largely similar analysis.

The present invention includes bispecific antibodies and antigen-binding fragments thereof that bind human CD3 with high affinity. The present invention also includes bispecific antibodies and antigen-binding fragments thereof that bind human CD3 with moderate or low affinity, depending on the therapeutic context and the specific targeting properties desired. For example, in the context of a bispecific antigen-binding molecule in which one arm binds CD3 and the other arm binds the target antigen (e.g.,

- 28 039429

CD20), it may be desirable that the target antigen binding arm bind the target antigen with high affinity, while the anti-CD3 arm binds CD3 with moderate or low affinity. In this way, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved, while avoiding general/untargeted CD3 binding and associated subsequent adverse side effects.

The present invention also includes antibodies and antigen-binding fragments thereof that bind human CD3 and induce T cell-mediated killing of tumor cells. For example, the present invention includes anti-CD3xCD20 antibodies that induce T cell mediated tumor cell killing with an EC50 of less than about 60 pM as measured by an in vitro T cell mediated tumor cell killing assay, e.g., the assay format described in Example 5 of this document. (e.g., assessments of the degree of killing of Raji tumor cells by human PBMCs in the presence of anti-CD3 antibodies), or a largely similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention induce T cell-mediated killing of tumor cells (e.g., PBMC-mediated killing of Raji cells) with an EC ₅₀ value of less than about 56 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM, as measured by an in vitro T cell-mediated tumor cell killing assay, eg, assay format described in Example 5 of this document, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding fragments thereof that bind human CD3/CD20 and induce complement dependent cytotoxicity (CDC), albeit to a lesser extent than antibodies having a wild type IgG Fc domain. For example, the present invention includes anti-CD3xCD20 antibodies that induce CSC killing of Raji or Daudi (DC20-expressing) cells with a percent cytotoxicity (%cytotoxicity) of less than about 50% as measured by an in vitro T-cell-mediated tumor cell killing assay, e.g., the assay format described in Example 6 of this document (e.g., target cell kill rates (Raji or Daudi) in the presence of complement and anti-CD3xCD20 antibodies), or a substantially similar assay. In certain embodiments, the antibodies or antigen-binding fragments of the present invention induce CSC cell killing (e.g., complement-mediated killing of Raji or Daudi cells) with a cytotoxicity percentage of less than about 45%, less than about 40%, less than about 35%. , less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less or no detectable background cytotoxicity cytotoxicity as measured by an in vitro complement-mediated cell killing assay, such as the assay format described in Example 6 of this document, or a substantially similar assay.

The present invention also includes antibodies and antigen-binding fragments thereof that bind human CD3/CD20 and do not significantly induce antibody-dependent cell-mediated cytotoxicity (ADCC) compared to antibodies having a wild type IgG Fc domain.

For example, the present invention includes anti-CD3xCD20 antibodies with unmeasurable killing of Raji or Daudi (CD20-expressing) cells with a percent cytotoxicity (%cytotoxicity) of less than about 20% (or less than background cytotoxicity) as measured by an in vitro NK cell mediated assay. cell killing, e.g., assay format described in Example 7 of this document (e.g., target cell kill rates (Raji or Daudi) in the presence of NK cells and anti-CD3xCD20 antibodies), or a substantially similar assay. Substantially similar assays may include the presence of NK cells, macrophages, neutrophils, eosinophils, or other FcγRIII-expressing cells, including cells expressing variant FcyRIII. In certain embodiments, the antibodies or antigen-binding fragments of the present invention exhibit no detectable ADCC activity (e.g., NK cell or FcyRIII mediated killing of Raji or Daudi cells) with a cytotoxicity percentage of less than about 30%, less than about 25%, less than less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less background cytotoxicity or no detectable cytotoxic activity, as measured by an in vitro FcyRIII-mediated cell killing assay, for example, the assay format described in Example 7 of this document, or a substantially similar assay.

In accordance with certain embodiments, the present invention includes antibodies and antigen-binding antibody fragments that bind human FcyRIIA (e.g., at 25° C.) with a CD of less than about 23.5 μM as measured by surface plasmon resonance, e.g. using the analysis format described in Example 8 of this document. In certain embodiments, antibodies or antigen-binding fragments of the present invention bind FcyRIIA with a Kd of less than about 20.5 μM, less than about 20 μM, less than about 19.3 μM, less than about 18 μM, less than about 17 µM, less than about 16 µM, less than about 15 µM, less than about 10 µM, less than about 9 µM, less than about 8 µM, less than about 7 µM, less than about 6 µM, less than about 5 µM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, or less than about 700 nM, as measured by surface plasmon resonance, for example , using the assay format described in Example 8 of this document (eg, mAb capture or antigen capture format), or a substantially similar assay.

In accordance with certain embodiments, the present invention includes antibodies and antigen-binding antibody fragments that bind human FcyRIIB (e.g., at 25° C.) with a Kd of less than about 233 μM as measured by surface plasmon resonance, e.g., using the assay format described in example 8 of this document. In certain embodiments, antibodies or antigen-binding fragments of the present invention bind FcyRIIB with a Kd of less than about 200 μM, less than about 190 μM, less than about 180 μM, less than about 170 μM, less than about 160 μM, less than about 150 µM, less than about 140 µM, less than about 130 µM, less than about 125 µM, less than about 123 µM, less than about 120 µM, less than about 110 µM, less than about 100 µM, less than about 90 μM, less than about 80 μM, less than about 70 μM, less than about 60 μM, less than about 50 μM, or less than about 40 μM as measured by surface plasmon resonance, e.g., using the assay format described in Example 8 of this document (eg, mAb capture or antigen capture format), or a substantially similar assay.

The present invention also includes antibodies and antigen-binding fragments that bind CD3xCD20 with a dissociative half-life (t1/ ₂ ) of greater than about 8 days as measured by surface plasmon resonance at 25 or 37°C, for example using the assay format described in example 9 of this document, or a largely similar analysis. In certain embodiments, the antibodies exhibit a t1/ ₂ of greater than about 5 days, greater than 6 days, greater than about 7 days, greater than about 8 days, greater than about 9 days, greater than about 10 days, greater than about 11 days, more than about 12 days, more than about 13 days, more than about 14 days, more than about 15 days, or more than about 20 days, as measured by surface plasmon resonance at 25 or 37°C, for example, using the assay format described in Example 9 of this document (eg, mAb capture or antigen capture format), or a substantially similar assay.

The present invention also includes anti-CD3/anti-CD20 bispecific antigen-binding molecules that do not exhibit significant activity in one or more assays selected from the group consisting of: (a) inducing proliferation of PBMCs in vitro; (b) CSC cytotoxicity (see, for example, example 6); (d) ADCC (see example 7 for example).

The present invention also includes anti-CD3/anti-CD20 bispecific antigen-binding molecules that exhibit significant activity in one or more assays selected from the group consisting of: (a) reducing the number of B cells (e.g., CD45+/CD20+ B cells) in cynomolgus monkeys (see, for example, examples 10 and 11); (b) reduction in B-cell tumor volume (eg, Raji tumor volume) in immunodeficient mouse models (see, for example, Example 12A) and (c) tumor regression in advanced tumor mouse models (see, for example, Example 12B) .

The present invention includes anti-CD3/anti-CD20 bispecific antigen-binding molecules that are capable of reducing the number of B cells in a subject (see, for example, example 10). For example, in accordance with certain embodiments of the invention, anti-CD3/antiCD20 bispecific antigen-binding molecules are provided, wherein a single administration of the bispecific antigen-binding molecule to a subject (e.g., at a dose of about 1, about 0.9, about 0.8 g, about 0, 7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, about 0.08, about 0.06 about 0.04, about 0.03 , about 0.02, about 0.01 mg/kg or less) results in a decrease in the number of B cells in the subject (eg, in a blood sample taken from the subject) below detectable levels. In certain embodiments of the invention, a single administration of an anti-CD3/anti-CD20 bispecific antigen-binding molecule at a dose of about 0.1 mg/kg results in a decrease in the number of B cells in a subject below detectable levels by about day 7, by about day 6 , about day 5, about day 4, about day 3, about day 2, or about day 1 after administration of the bispecific antigen-binding molecule to a subject. In accordance with certain embodiments of the invention, a single administration of an anti-CD3/anti-CD20 bispecific antigen-binding molecule in accordance with the invention at a dose of about 0.01

- 30 039429 mg/kg, causes the number of B cells to remain below detectable levels for at least about 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17 days or more after introductions. In the context of this document, the expression below detectable levels means that the presence of B-cells cannot be directly or indirectly determined in a blood sample taken from a subject using standard detection methods, for example, the FACS analysis for B-cell markers given in example 10 of this document.

Alternatively, one first dose is administered at a low dosage, such as 10 or 30, or 100 μg, and then after some time a second dose is administered at a higher dosage, for example, two or three times the first dosage, to prevent, reduce or soften the cytokine storm in the patient's body. By reducing the cytokine storm in the patient's body is meant a reduction in the action of the cytokine cascade or hypercytokinemia, and such a negative immune response can be caused, without limitation, by a positive feedback between cytokines and white blood cells and/or highly elevated levels of various cytokines.

In accordance with example 20, a first (initial) dose of a bespecifically antigen-binding molecule according to the invention (eg, Ab1) is administered, followed by a second dose some time later, with the second dose exceeding (being greater than) the first dose. In some embodiments, the second dose is about 2 times or about 3 times the first dose. In another embodiment, the bispecific antigen binding molecule is administered weekly as a first dose for consecutive weeks, eg four (4) consecutive weeks. In another embodiment of the invention, the first dose is administered weekly, followed by monthly doses for an additional period of time (or with a certain number of monthly doses). In some embodiments of the invention, after a certain dosing regimen of the first dose, the second dose is administered weekly followed by monthly doses for an additional period of time. In some embodiments, the first (initial) dose is 10 μg and the second dose is 30 μg. In some embodiments, the first (initial) dose is 30 μg and the second dose is 100 μg. In other embodiments, the first (initial) dose is 100 μg and the second dose is 300 μg. In other embodiments, the first (initial) dose is 300 μg and the second dose is 100 μg. In other embodiments, the first (initial) dose is 1000 μg and the second dose is 2000 μg. In other embodiments, the first (initial) dose is 2000 μg and the second dose is 3000 μg. In other embodiments of the invention, the first (initial) dose is 3000 μg and the second dose is 4000 μg. In other embodiments of the invention, the first (initial) dose is 4000 μg and the second dose is 5000 μg. In other embodiments, the first (initial) dose is 5000 μg and the second dose is 6000 μg. In other embodiments, the first (initial) dose is 6000 μg and the second dose is 7000 μg. In other embodiments, the first (initial) dose is 7000 μg and the second dose is 8000 μg.

The present invention also provides anti-CD3/anti-CD20 bispecific antigen-binding molecules that, when administered to a subject, result in no more than a temporary decrease in the number of T cells. For example, anti-CD3/anti-CD20 bispecific antigen-binding molecules are provided which, when administered to a subject at a dose of about 0.01, or about 0.1 mg/kg, or about 1 mg/kg, result in a decrease in the number of T cells. 1 day after administration, after which the number of T cells per microliter of blood is restored (for example, approximately 2, 4, 7, 14, 28 days or later after administration). For example, the present invention provides an anti-CD3/anti-CD20 bispecific antigen-binding molecule, wherein the number of T cells per microliter of blood taken from a subject approximately 4-7 days after administration of the antigen-binding molecule at a dose of about 0.01 or about 0 .1 mg/kg, or about 1 mg/kg, is equal to or greater than the number of T cells per microliter of blood drawn from the subject prior to administration of the antigen-binding molecule, as determined by standard T cell detection methods, such as the FACS assay for T cells. markers shown in example 10 of this document.

Epitope mapping and related techniques

The epitope on CD3 or CD20 to which the antigen-binding molecules of the present invention bind may consist of one contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the CD3 protein. Alternatively, an epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of CD3 or CD20. Antibodies of the invention may interact with amino acids from one CD3 chain (eg CD3 epsilon, CD3 delta or CD3 gamma) or may interact with amino acids on two or more different CD3 chains. As used herein, the term epitope refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. One antigen may have more than one epitope. Thus, different antibodies can bind

- 31 039429 with different sites on the antigen and may have different biological effects. Epitopes may be conformational or linear. A conformational epitope is formed by spatially combined amino acids from different segments of a linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. Under certain conditions, an epitope on an antigen may contain saccharide components, phosphoryl groups, or sulfonyl groups.

Those skilled in the art are aware of various techniques that can be used to determine if the antigen-binding domain of an antibody interacts with one or more amino acids in a polypeptide or protein. Exemplary techniques include, for example, the standard cross-blocking assay described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation assay, peptide blot assay (Reineke, 2004, Methods Mol Biol 248 :443-463) and peptide digestion analysis. In addition, techniques such as epitope exclusion, epitope removal, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to determine the amino acids in a polypeptide that the antigen-binding domain of an antibody interacts with is hydrogen-deuterium exchange with mass spectrometric detection. In general terms, the hydrogen-deuterium exchange method comprises deuterium labeling of a protein of interest, followed by binding of an antibody to the deuterium labeled protein. The protein/antibody complex is then transferred to water to allow hydrogen-deuterium exchange in all residues except for the residues protected by the antibody (which remain labeled with deuterium). After dissociation of the antibody, the target protein is subjected to protease digestion and mass spectrometric analysis, thus revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73: 256A-265A. X-ray diffraction crystallography of the antigen/antibody complex can also be used for epitope mapping purposes.

The present invention further includes anti-CD3 antibodies that bind to the same epitope as any of the specific generic antibodies described herein (eg, antibodies containing any of the amino acid sequences shown in Table 1). Similarly, the present invention also includes anti-CD3 antibodies that compete for CD3 binding with any of the specific generic antibodies described herein (eg, antibodies containing any of the amino acid sequences shown in Table 1).

The present invention also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3 and a second antigen-binding domain that specifically binds human CD20, wherein the first antigen-binding domain binds to the same epitope on CD3 as specific exemplary CD3-specific antigen-binding domains, described herein and/or the second antigen-binding domain binds to the same epitope on CD20 as the particular exemplary CD20-specific antigen-binding domains described herein.

Similarly, the present invention also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds human CD3 and a second antigen-binding domain that specifically binds human CD20, wherein the first antigen-binding domain competes for CD3 binding with any of the specific exemplary CD3-specific antigen-binding domains described herein, and/or the second antigen-binding domain competes for binding to CD20 with any of the specific exemplary CD20-specific antigen-binding domains described herein.

Whether an antigen-binding molecule (eg, an antibody) or its antigen-binding domain binds to the same epitope or competes for binding with a standard antigen-binding molecule of the present invention can be easily determined using conventional methods known in the art. For example, to determine if an antibody of interest binds to the same epitope on CD3 (or CD20) as the standard bispecific antigen binding molecule of the present invention, the standard bispecific antigen binding molecule is allowed to bind to the CD3 protein (or CD20 protein). Next, the possibility of binding of the test antibody to the CD3 (or CD20) molecule is assessed. If the test antibody can bind to CD3 (or CD20) after saturating binding to a standard bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope on CD3 (or CD20) than the standard bispecific antigen-binding molecule. On the other hand, if the test antibody cannot bind to the CD3 (or CD20) molecule after saturating binding to the standard bispecific antigen binding molecule, then the test antibody can bind to the same CD3 (or CD20) epitope as the epitope bound by the standard bispecific antigen binding molecule. according to the invention. Additional routine experiments (e.g., peptide mutation and binding assay) can then be performed to confirm that the observed lack of binding of the antibody of interest is in fact due to

- 32 039429 binding to the same epitope as in the case of a standard bispecific antigen-binding molecule, or that steric blocking (or other phenomenon) is responsible for the lack of observed binding. Experiments of this type can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present invention, two antigen-binding proteins bind to the same (overlapping) epitope if, for example, a 1-, 5-, 10-, 20-, or 100-fold excess of one antigen-binding protein inhibits binding of the other at least at least 50%, but preferably 75%, 90% or even 99% according to competitive binding assays (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antigen-binding proteins are said to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antigen-binding protein reduce or eliminate the binding of the other. Two antigen-binding proteins are considered to have overlapping epitopes unless a subset of amino acid mutations that reduces or eliminates the binding of one antigen-binding protein reduces or eliminates the binding of another.

To determine if an antibody or its antigen-binding domain competes for binding to a standard antigen-binding molecule, the binding experiment described above is performed in two orientations: antibodies with a CD3 (or CD20) molecule. In the second orientation, the test antibody is allowed to bind to the CD3 (or CD20) molecule under saturating conditions, followed by evaluation of the binding of the standard antigen-binding molecule to the CD3 (or CD20) molecule. If in both orientations only the first (saturating) antigen-binding molecule is able to bind to the CD3 (or CD20) molecule, it can be concluded that the antibody under test and the standard antigen-binding molecule compete for binding to CD3 (or CD20). One of skill in the art will appreciate that an antibody that competes for binding to a standard antigen-binding molecule may not necessarily bind to the same epitope as the standard antibody, but may sterically block the binding of the standard antibody by binding to an overlapping or contiguous epitope.

Preparation of antigen-binding domains and construction of bispecific molecules

Antigen-binding domains specific for particular antigens can be generated using any antibody generation technique known in the art. Once two different antigen-binding domains specific for two different antigens (eg, CD3 and CD20) have been obtained, they can be properly ordered relative to each other to obtain a bispecific antigen-binding molecule of the present invention using standard methods.

(A discussion of exemplary bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules of the present invention is provided elsewhere in this document). In certain embodiments of the invention, one or more individual components (eg, heavy and light chains) of the multispecific antigen-binding molecules of the invention are derived from chimeric, humanized, and fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and/or light chains of the bispecific antigen-binding molecules of the present invention can be produced using the VELOCIMMUNE™ technology. When using VELOCIMMUNE™ technology (or any other human antibody technology), high affinity chimeric antibodies for a specific antigen (eg, CD3 or CD20) are first isolated, having a human variable region and a mouse constant region. Antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. Mouse constant regions are substituted with the desired human constant region to produce fully human heavy and/or light chains that can be incorporated into the bispecific antigen binding molecules of the present invention.

Genetically engineered animals can be used to create human bispecific antigen-binding molecules. For example, a genetically modified mouse that is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence can be used, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant region gene in endogenous mouse kappa locus. Such a genetically modified mouse can be used to generate fully human bispecific antigen binding molecules containing two different heavy chains that are linked to an identical light chain containing a variable domain derived from one or more human light chain variable region gene segments. (See, for example, US 2011/0195454 for a detailed discussion of such engineered mice and their use in the production of bispecific antigen-binding molecules).

Bioequivalents

The present invention includes antigen-binding molecules having amino acid sequences that differ from the typical molecules disclosed herein, but retain the ability to bind CD3 and/or CD20. Such variant molecules may contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit biological activity that is substantially equivalent to that of the bispecific antigen-binding molecules described.

The present invention includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered to be bioequivalent if, for example, they are pharmaceutical equivalents or alternatives whose rate and extent of absorption does not show a significant difference when administered at the same molar dose and under similar conditions, either as a single dose or multiple doses. Some antigen-binding proteins are considered equivalent or pharmaceutically alternative if they are equivalent in absorption but not in absorption rate, but may be considered bioequivalent because such a difference in absorption rate is intentional and reflected in the labeling, and is also not essential to achieve effective drug concentrations. drugs in the body, such as chronic use, and is considered medically insignificant for the particular investigational medicinal product.

In one embodiment, two antigen-binding proteins are bioequivalent if there is no clinically significant difference in their safety, purification, and efficacy.

In one embodiment of the invention, two antigen-binding proteins are bioequivalent if the patient can switch one or more times between a standard product and a biological product without an expected increase in the risk of adverse events, including a clinically significant change in immunogenicity or a decrease in efficacy compared to continuing therapy without such a switch.

In one embodiment of the invention, two antigen-binding proteins are bioequivalent if they both act according to a common mechanism or mechanisms of action in relation to the corresponding pathological condition or conditions, to the extent that such mechanisms are known.

Bioequivalence can be demonstrated using in vivo and in vitro methods. The determination of bioequivalence includes, for example, (a) an in vivo study in humans or other mammals, which measures the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro study that has been correlated and is reasonably predictive in humans of in vivo bioavailability; (c) an in vivo study in humans or other mammals, which determines the corresponding short-term pharmacological effect of the antibody (or its target) as a function of time; and (d) a well-controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of the antigen-binding protein.

Bioequivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by creating various residue or sequence substitutions, or deleting terminal or internal residues or sequences unnecessary for biological activity. For example, cysteine residues not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges after renaturation. In another context, bioequivalent antigen-binding proteins may include variants of the bispecific antigen-binding molecules described herein containing amino acid changes that modify the glycosylation characteristics of the molecules, such as mutations that abrogate glycosylation.

Species selectivity and species cross-reactivity

According to certain embodiments of the invention, antigen-binding molecules are provided that bind to human CD3 but not CD3 of other species. Also provided are antigen-binding molecules that bind to human CD20 but not CD20 of other species. The present invention also includes antigen-binding molecules that bind to human CD3 and CD3 of one or more non-human species; and/or antigen-binding molecules that bind to human CD20 and CD20 of one or more non-human species.

In accordance with certain exemplary embodiments of the invention, antigen-binding molecules are provided that bind to human CD3 and/or human CD20 and may or may not bind, as the case may be, to CD3 and/or CD20 of one or more mouse species. , rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee. For example, in a particular exemplary embodiment, the present invention provides bispecific antigen-binding molecules comprising a first antigen-binding domain that binds human CD3 and cynomolgus CD3 and a second antigen-binding domain that specifically binds human CD20.

Immunoconjugates

The present invention includes antigen-binding molecules conjugated to a therapeutic moiety (immunoconjugate), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Cytotoxic agents include any agents that can harm a cell. Examples of suitable cytotoxic agents for the formation of immunoconjugates are known in the art (see, for example, WO 05/103081).

Therapeutic formulations and administration

As used herein, the terms effective amount and therapeutically effective amount refer to an amount of active therapeutic agent sufficient to produce the desired therapeutic response without undue adverse side effects such as toxicity, irritation, or allergic reaction. The precise effective amount will obviously vary depending on such factors as the particular condition being treated, the physical condition of the patient, the type of animal being treated, the duration of treatment, the nature of the concomitant therapy (if any) and the specific formulations employed. as well as the structure of compounds or their derivatives. In such a case, an amount is considered to be therapeutically effective if it results in, but is not limited to, one or more of the following: (a) inhibition of tumor growth (eg, B-cell cancer); and (b) extinction or stabilization of B-cell cancer.

The dose of antigen-binding molecule administered to a patient may vary depending on the age and size of the patient, the target disease, conditions, route of administration, and the like. The preferred dose is generally calculated according to body weight or body surface area. When the bispecific antigen-binding molecule of the present invention is administered therapeutically to an adult patient, it may be appropriate to intravenously administer the bispecific antigen-binding molecule of the present invention, typically in a single dose of about 0.01 to about 20 mg/kg body weight, greater than preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg of body weight. Depending on the severity of the pathological condition, the frequency and duration of treatment can be adjusted. Effective dosages and regimens for administering the bispecific antigen-binding molecule can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation and the dose adjusted accordingly. In addition, cross-species dosing can be done using methods well known in the art (eg, Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

A large number of different delivery systems are known that can be used to administer the pharmaceutical composition of the invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987 , J Biol Chem 262:4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through the epithelial or mucocutaneous lining (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can also be administered together with other biologically active agents. The introduction can be systemic or local.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, in the case of subcutaneous delivery, a pen-type device can be used to deliver the pharmaceutical composition of the present invention. Such a pen-type device may be reusable or disposable. A refillable pen device typically uses a replaceable cartridge that contains a pharmaceutical composition. After all of the pharmaceutical composition contained in the cartridge has been introduced and the cartridge has been emptied, the empty cartridge can be easily removed and replaced with a new cartridge which contains the pharmaceutical composition. The pen device can then be reused. There is no replaceable cartridge in a disposable pen-type device. A disposable pen device is supplied pre-filled with a pharmaceutical composition in a reservoir inside the device. Once the reservoir has run out of pharmaceutical composition, dispose of the entire device.

Numerous reusable delivery devices such as pens and auto-injectors

- 35 039429 used for subcutaneous delivery of the pharmaceutical composition according to the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, pen HUMALOG™, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark) , BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen type delivery devices used for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly ), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, L.P.), and HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name a few. .

In certain instances, the pharmaceutical composition may be delivered via a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment of the invention, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In another embodiment, the controlled release system can be placed near the target of the composition so that only a fraction of the systemic dose is required (see e.g. Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 ). Other controlled release systems are reviewed by Langer, 1990, Science 249:1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectables can be prepared by conventional methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending, or emulsifying an antibody as described above, or a salt thereof, in a sterile aqueous or oily medium conventionally used for injection. As the aqueous injection medium, there can be mentioned, for example, physiological saline, isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with a suitable solubilizing agent such as alcohol (for example, ethanol), polyhydric alcohol (eg, propylene glycol, polyethylene glycol), non-ionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)], etc. As the oil medium, for example, sesame oil, soybean oil, etc. are used, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into a suitable ampoule.

Preferably, the above-described pharmaceutical compositions for oral or parenteral use are formulated as unit dose dosage forms corresponding to the dosage of the active ingredients. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (eg, ampoules, vials), suppositories, and the like.

Therapeutic applications of antigen-binding molecules

The present invention includes methods comprising administering to a subject in need thereof a therapeutic composition comprising an anti-CD3 antibody or a bispecific antigen-binding molecule that specifically binds CD3 and a target antigen (eg, CD20). The therapeutic composition may contain any antibodies or bispecific antigen-binding molecules disclosed herein and a pharmaceutically acceptable carrier or diluent. In the context of this document, the term in need of it means a human or non-human animal that exhibits one or more symptoms or signs of cancer (for example, a subject expressing a tumor or suffering from any of the following types of cancer), or for which any or otherwise, inhibition or reduction of CD20 activity or reduction in the number of CD20+ B cells or regression of CD20+ B cell tumors would be beneficial.

The antibodies and bispecific antigen-binding molecules of the invention (and therapeutic compositions containing them) are useful, among other things, in the treatment of any disease or disorder in which stimulating, activating and/or targeting an immune response would be beneficial. In particular, the anti-CD3/anti-CD20 bispecific antigen binding molecules of the present invention can be used to treat, prevent and/or reduce any disease or disorder associated with or mediated by CD20 expression or activity or CD20+ B cell proliferation. The mechanism of action by which the therapeutic methods of the invention are carried out involves killing cells expressing CD20 in the presence of effector cells, for example, by apoptosis, phagocytosis, or

- 36 039429 combinations of two or more of these mechanisms or similar cytotoxic mechanisms. CD20-expressing cells that can be inhibited or killed with the bispecific antigen-binding molecules of the invention include, for example, carcinogenic B cells.

Tumor burden reduction or tumor regression includes partial or complete disappearance of a tumor or tumors in a subject. It is understood that tumor regression is a trend towards a reduction in tumor burden or a less severe disease state. Therefore, regression is a progressive decrease in detectable malignancies in the body, including a decrease in tumor size and/or a decrease in the number of tumors. Reducing tumor development includes partial or complete inhibition or suppression of additional or new tumor growth.

The antigen binding molecules of the present invention can be used to treat, for example, primary and/or metastatic tumors arising in the brain and meninges, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscles, bones, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the bispecific antigen-binding molecules of the invention are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, breast cancer, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, cancer stomach (eg, gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, or melanoma. In accordance with certain exemplary embodiments of the invention, the bispecific antigen-binding molecules of the present invention are used to treat B-cell cancers (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], lymphoblastic leukemia/progenitor B-cell lymphoma, mature B-cell neoplasms , chronic B-cell lymphocytic leukemia/lymphoma/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicle center cell lymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell cellular lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström's macroglobulinemia, and anaplastic large cell lymphoma).

The anti-CD3/anti-CD20 bispecific antigen-binding molecules of the present invention are administered in an amount sufficient to reduce tumor burden, promote tumor regression, inhibit tumor growth, or reduce tumor development in a subject. In some exemplary embodiments of the invention, the amount administered is from about 0.001 to about 1 mg/kg. In other embodiments, the amount administered is about 0.4 mg/kg. In other embodiments, the amount administered is about 0.04 mg/kg. In other embodiments, the amount administered is about 0.004 mg/kg.

In accordance with certain embodiments of the present invention, antigen-binding molecules are used to treat a patient afflicted with a B-cell lymphoma (eg, NHL) that is resistant or not fully responsive to anti-CD20 therapy alone (eg, resistant to rituximab therapy). According to other related embodiments, methods are provided comprising administering an anti-CD3/antiCD20 bispecific antigen-binding molecule disclosed herein to a patient afflicted with B-cell lymphoma (e.g., NHL) that is refractory to anti-CD20 therapy (e.g., rituximab-refractory tumor or relapsed or refractory B-cell lymphoma). Analytical/diagnostic methods known in the art, such as tumor scanning, etc., can be used to ensure that a patient has a tumor that is resistant or not completely responsive or refractory to anti-CD20 therapy alone.

The present invention also includes methods for treating residual cancer in a subject. In the context of this document, the term residual cancer means the presence or persistence of one or more cancer cells in the body of a subject after treatment with anticancer therapy.

In accordance with certain aspects, the present invention provides methods for treating a disease or disorder associated with CD20 expression (e.g., B-cell lymphoma), comprising administering to a subject one or more of the bispecific antigen-binding molecules described elsewhere herein, after the subject has has undergone anti-CD20 monotherapy (for example, after administration of a pharmaceutical composition containing an anti-CD20 antibody, such as rituximab). For example, the present invention includes methods for treating B-cell lymphoma comprising administering an anti-CD3/anti-CD20 bispecific antigen-binding molecule to a patient after 1, 2, 3, 4, 5, 6 days, 1, 2, 3, or 4 weeks, 2, 4, 6, 8 months, 1 year or more after the subject received anti-CD20 monotherapy (eg, treatment with rituximab or treatment with its equivalent).

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Combination therapy and formulations

The present invention provides methods that include administering a pharmaceutical composition comprising any of the generic antibodies and bispecific antigen-binding molecules described herein in combination with one or more additional therapeutic agents. Typical additional therapeutic agents that can be combined or administered in combination with an antigen binding molecule of the present invention include, for example, an EGFR antagonist (eg, an anti-EGFR antibody [eg, cetuximab or panitumumab] or a small molecule EGFR inhibitor [eg, gefinitib or erlotinib ]), an antagonist of another member of the EGFR family such as Her2/ErbB2, ErbB3, or ErbB4 (e.g., an anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), an EGFRvIII antagonist (e.g., an antibody that specifically binds EGFRvIII), cMET antagonist (eg, anti-cMET antibody), IGF1R antagonist (eg, anti-IGF1R antibody), B-raf inhibitor (eg, vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR- inhibitor α (eg, anti-PDGFR-a antibody), PDGFR-β inhibitor (eg, anti-PDGFR-e antibody), VEGF antagonist (eg, VEGF-Trap, see, for example, US 7,087,411 (also referred to as and herein a VEGF inhibitory fusion protein), an anti-VEGF antibody (eg, bevacizumab), a small molecule VEGF receptor kinase inhibitor (eg, sunitinib, sorafenib, or pazopanib)), a DLL4 antagonist (eg, an anti-DLL4 antibody disclosed in US 2009 /0142354), an Ang2 antagonist (for example, an anti-Ang2 antibody disclosed in US 2011/0027286, such as H1H685P), a FOLH1 antagonist (for example, an anti-FOLHl antibody), a PRLR antagonist (for example, an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist (for example , anti-STEAP1 antibody, or anti-STEAP2 antibody), TMPRSS2 antagonist (eg, anti-TMPRSS2 antibody), MSLN antagonist (eg, anti-MSLN antibody), CA9 antagonist (eg, anti-CA9 antibody), uroplakin antagonist (eg, anti- uroplakin), a monovalent CD20 antagonist (eg, a monovalent anti-CD20 antibody such as rituximab), etc. Other agents that are advantageous when administered in combination with the antigen binding molecules of the invention include cytokine inhibitors, including small molecule cytokine inhibitors, and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors. Pharmaceutical compositions of the present invention (e.g., pharmaceutical compositions comprising the anti-CD3/anti-CD20 bispecific antigen-binding molecule disclosed herein) may also be administered as part of a therapeutic regimen involving the use of one or more therapeutic combinations selected from ICE: ifosfamide (eg, Ifex®), carboplatin (eg, Paraplatin®), etoposide (eg, Etopophos®, Toposar®, Vepezid®, VP-16); DHAP: dexamethasone (eg Decadron®), cytarabine (eg Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (eg Platinol®-AQ); and ESHAP: etoposide (eg Etopophos®, Toposar®, Vepezid®, VP-16), methylprednisolone (eg Medrol®), high dose cytarabine, cisplatin (eg Platinol®-AQ).

The present invention also includes therapeutic combinations containing any of the antigen binding molecules specified herein and an inhibitor of one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII, cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1, PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the above cytokines, wherein the inhibitor is an aptamer, antisense, ribozyme, siRNA, peptibody, nanobody, or antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other engineered molecules such as diabodies, tribodies, minibodies, and minimal recognition components).

The antigen-binding molecules of the invention can also be administered and/or mixed in combination with antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs. The antigen-binding molecules of the invention can also be administered as part of a treatment regimen that also includes radiation therapy and/or conventional chemotherapy.

Additional therapeutically active components can be administered immediately before, simultaneously with or shortly after administration of the antigen-binding molecule according to the present invention (for the purposes of the present invention, such administration regimens are considered to be the administration of the antigen-binding molecule in combination with an additional therapeutically active component).

The present invention includes pharmaceutical compositions in which the antigen-binding molecule of the present invention is mixed with one or more additional therapeutically active ingredients as described elsewhere herein.

Introduction schemes

In accordance with certain embodiments of the present invention, multiple doses of an antigen-binding molecule (eg, an anti-CD3 antibody or a bispecific antigen-binding molecule that specifically binds CD20 and CD3) may be administered to a subject over a course of time. Methods in accordance with this aspect of the invention include sequential

- 38 039429 introduction to the subject of multiple doses of the antigen-binding molecule according to the invention. As used herein, sequential administration means that each dose of an antigen-binding molecule is administered to a subject at different points in time, eg on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient one initial dose of antigen-binding molecule followed by one or more secondary doses of antigen-binding molecule and optionally one or more tertiary doses of antigen-binding molecule.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of the antigen-binding molecule according to the invention. Thus, the initial dose or first dose is the dose that is administered at the beginning of the treatment regimen (also called the initial dose); secondary doses are doses that are administered after the initial dose; and tertiary doses are doses administered after secondary doses. Initial, secondary and tertiary doses may contain the same amount of antigen-binding molecule, but in General may differ from each other in terms of frequency of administration. However, in certain embodiments of the invention, the amount of antigen-binding molecule contained in the initial, secondary and / or tertiary doses differs from each other (for example, adjusted up or down depending on the situation) during the course of treatment. In certain embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of a treatment regimen as loading doses, followed by doses administered at a lesser frequency (eg, maintenance doses).

It has been found that administration of a first dose administered at a minimum concentration followed by a subsequent or second dose administered at two or three times the first dose will have a beneficial effect on the patient.

In some embodiments, a first dose of an antigen-binding molecule is administered to a patient sequentially over a first period of time, and a subsequent second dose of said antigen-binding molecule is administered to a patient sequentially over a second period of time, wherein said second dose is greater than said first dose. In other embodiments, the first dose is administered weekly or twice weekly for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. In another embodiment, the second dose is administered once a week, twice a week, once a month or twice a month for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks or months.

In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 1-26 (for example, 1, 11/2, ₂ , 21/2, 3, 31/2, ₄ , 41/2, 5, 51/ ₂ , 6, 6 ^1/2 , 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2 , 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22 , 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2 or more) weeks after the immediately previous dose. In the context of this document, the expression immediately previous dose means, in the case of a sequence of multiple injections, the dose of antigen-binding molecule that is administered to the patient before the administration of the immediately next dose in the sequence, without any intermediate doses.

Methods in accordance with this aspect of the invention may include administering multiple secondary and/or tertiary doses of an antigen-binding molecule (eg, a bispecific antigen-binding molecule that specifically binds CD20 and CD3) to a patient. For example, in certain embodiments of the invention, only one secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments of the invention, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

In embodiments of the invention involving the use of multiple secondary doses, each secondary dose can be administered at the same frequency as the remaining secondary doses.

For example, each secondary dose may be administered to a patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments of the invention involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the remaining tertiary doses. For example, each tertiary dose may be administered to a patient 2-4 weeks after the immediately previous dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to a patient may vary during the course of a treatment regimen. The frequency of administration can also be adjusted by the attending physician during the course of treatment, depending on the needs of the individual patient after clinical examination.

Diagnostic applications of antibodies

The Ahtu-CD3xCD20 antibodies of the present invention can also be used to detect and/or detect CD3 or CD3 expressing cells in a sample, for example for diagnostic purposes. For example, an anti-CD3xCD20 antibody or fragment thereof can be used to diagnose a pathological condition characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of CD3. Typical diagnostic methods of analysis

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CD3 may include, for example, contacting a patient-derived sample with an anti-CD3xCD20 antibody of the invention, the antibody labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-CD3xCD20 antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself labeled with a detectable label. The detectable label or reporter molecule may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I; a fluorescent or chemiluminescent substance such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assay methods that can be used to detect or determine CD3 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS).

The Ahtu-CD3xCD20 antibodies of the present invention can also be used to detect and/or detect CD20 or CD20 expressing cells in a sample, or similarly, to diagnose a pathological condition or disease characterized by aberrant expression (e.g., overexpression, underexpression, lack of expression, etc.) CD20.

Samples that can be used in the CD3 or CD20 diagnostic assays of the present invention include a tissue or fluid sample obtained from a patient that contains detectable amounts of CD3 and/or CD20 proteins or fragments thereof under normal or pathological conditions. In general, CD3 or CD20 levels are first measured in a specific sample from a healthy patient (e.g., a patient not affected by a disease or condition associated with abnormal CD3 or CD20 levels or activity) to establish a baseline or standard CD3 or CD20 level. This baseline CD3 or CD20 level can then be compared with CD3 levels measured in samples obtained from individuals suspected of having a disease or condition associated with CD3 or CD20, respectively.

Examples

The following examples are provided to provide those skilled in the art with a full disclosure and description of how to make and use the methods and compositions of the invention, and do not limit the scope of what the authors consider their invention. Care has been taken to maintain accuracy with respect to the numbers used (eg quantities, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, fractions are in parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1 Generation of Anti-CD3 and Anti-CD20 Antibodies

Several methods are known for isolating anti-CD3 or anti-CD20 antibodies. The fully human anti-CD3 antibodies used in the following examples were isolated directly from antigen-positive B cells without fusion with myeloma cells, as described in US 2007/0280945A1.

Additional examples of anti-CD3 antibodies can be used in these methods, and a description of such antibodies and the biological properties of such anti-CD3 antibodies are described in PCT International Application No. PCT/US13/60511, filed September 19, 2013, which is incorporated in its entirety in this document by reference. Exemplary anti-CD20 antibodies and the biological properties of such anti-CD20 antibodies are described in US 7,879,984 and PCT International Application No. PCT/US13/60511, filed September 19, 2013, which are incorporated herein by reference.

The Ahtu-CD20 antibody and antibody construction method used to construct the bispecific antibodies in this example are as described in US 7,879,984.

The amino acid designations of the heavy and light chain variable regions and CDRs used to construct the anti-CD3 antigen-binding arm and anti-CD20 binding arm of the bispecific antibodies of the invention are shown in Table 1. 1. The corresponding designations of the nucleotide sequences are given in table. 2.

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Table 1. Amino acid sequence designations (SEQ ID NO)

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 Anti- CD2 0 2 four 6 eight eighteen twenty 22 24 Anti-POPs ten 12 fourteen 16 eighteen twenty 22 24

Table 2. Nucleotide sequence designations (SEQ ID NO)

SEQID NO: Antibody designation HCVR HCDR1 HCDR2 HDDR3 LCVR LCDR1 LCDR2 LCDR3 Anti- CD2 0 one 3 5 7 17 19 21 23 Ahth-CD3 9 eleven 13 fifteen 17 19 21 23

Example 2 Generation of bispecific antibodies that bind CD3 and CD20

Bispecific antibodies were constructed containing an anti-COP-specific binding domain and an anti-CO20-specific binding domain with the above sequences using standard techniques in which the heavy chain and light chain from an anti-COP antibody were combined with the heavy chain from an anti-CO20 antibody. .

Thus, the bispecific antibodies constructed in accordance with the present example contain two separate antigen-binding domains (ie, binding arms). The first antigen binding domain comprises a heavy chain variable region derived from an anti-CO20 antibody (CD20-VH) coupled to a light chain variable region derived from an anti-COP antibody (CD3-VL). CD20-VH/CD3-VL pairing creates an antigen-binding domain that specifically recognizes CD20. The second antigen binding domain comprises a heavy chain variable region derived from an anti-COP antibody (CD3-VH) coupled to a light chain variable region derived from an anti-COP antibody (CD3-VL). CD3-VH/CD3-VL pairing creates an antigen-binding domain that specifically recognizes CD3. The same CD20-VH was used in all bispecific antibodies generated in this example and was designated CD20-VH-A.

The wild type heavy chain constant domain (CH) for each heavy chain was replaced with a chimeric CH using recombinant techniques. The CH of one binding arm (eg, the anti-COP binding arm) contains a mutation in the CH3 region of the CH that facilitates the isolation of the bispecific antibody.

Summarized data on the constituent parts of various bispecific antibodies, created in accordance with the example, are shown in table. 3 and 4.

Table 3. Amino acid sequence designations

Name of the bispecific antibody Ahth-CD20 antigen binding domain Anti-POP antigen binding domain Heavy chain variable region Heavy chain region constants Light chain variable region Heavy chain variable region Heavy chain constant region Light chain variable region CD20-VH-A CH CD3-VL-A CD3-VH-A CH CD3-VL-A Antibody 1 2 26 eighteen ten 28 eighteen Antibody 2 2 thirty eighteen ten 32 eighteen

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Table 4. Designations of nucleotide sequences

Name of the bispecific antibody Ahth-CD20 antigen binding domain Ahth-CD3 antigen binding domain Heavy chain variable region Heavy chain constant region Light chain variable region Heavy chain variable region The constant region of heavy chains Light chain variable region CD20-VH-A CH CD3-VL-A CD3-VH-A CH CD3-VL-A Antibody 1 one 25 17 9 27 17 Antibody 2 one 29 17 9 31 17

In table. 5 and 6 show the amino acid sequence designations for the various heavy chain variable regions (Table 5) and light chain variable regions (Table 6) with their respective CDRs for the bispecific antibodies in this example.

Table 5. Heavy chain amino acid sequence designations

Heavy chain designation Heavy chain variable region (HCVR) HCDR1 HCDR2 HDDR3 Heavy chain constant region (CH) CD20-VH-CH-A 2 four 6 eight 2 6 or 2 8 or 3 0 or 32 CD3-VH-CH-A ten 12 fourteen 16

Table 6. Light chain amino acid sequence designations

Light chain designation Light chain variable region (LCVR) LCDR1 LCDR2 LCDR3 CD3-VL-A eighteen twenty 22 24

In addition, in Table. 7 and 8 show the sequence designations for the nucleotide sequences encoding the various heavy chain variable regions (Table 7), heavy chain variable constant regions, and light chain variable regions (Table 8) with their respective CDRs for the bispecific antibodies in this example.

Table 7. Heavy chain nucleotide sequence designations

Heavy chain designation Heavy chain variable region (HCVR) HCDR1 HCDR2 HDDR3 Heavy chain constant region (HCVR) CD20-VH-A one 3 5 7 25 or 27 or 2 9 or 31 CD3-VH-A 9 eleven 13 fifteen

Table 8. Light chain nucleotide sequence designations

Light chain designation Varna white light chain region (LCVR) LCDR1 LCDR2 LCDR3 CD3-VL-A 17 19 21 23

In addition to the bispecific antibodies described above, the following control antibodies were used in certain experiments in the examples:

Control antibody 1: Monoclonal antibody OKT-3 against human T-cell surface antigens described in US 4361549 and available from hybridoma CRL-8001 (American

- 42 039429 type culture collection, Manassas, VA).

Control Antibody 2: Antibody SP34, reactive antibody against T3 complex epsilon chain on human T-lymphocyte cells, available from BD Pharmagen, Cat # 55052.

Control antibody 3: Ahtu-CD20 therapeutic antibody having the rituxan (rituximab) heavy and light chain sequences disclosed in US 5,736,137.

(Control) Antibody 4: Also known as CD3xCD20 wild-type Fc antibody (dtFc), this antibody was generated in analogy to the methods described above and has an anti-CD20 arm containing the amino acid sequence pair HCVR/LCVR SEQ ID NO: 2/18 ( amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 SEQ ID NO: 4-6-8-20-22-24) and wild-type IgG1 CH region (SEQ ID NO: 45); and an anti-CD3 arm containing a pair of amino acid sequences HCVR/LCVR SEQ ID NO: 10/18 (amino acid sequences HCDR1-HCDR2HCDR3-LCDR1-LCDR2-LCDR3 SEQ ID NO: 12-14-16-20-22-24) and CH wild-type IgG1 region with two amino acid modifications (SEQ ID NO: 48) in the CH3 domain to facilitate isolation. The CD3xCD20 antibody-dtFc (Antibody 4) may be referred to as a similar control antibody having a wild-type IgG1 Fc domain (i.e. similar to the antigen-binding domains of the CD3xCD20 antibodies chimeric Fc of the invention) in order to compare antibody effector functions or other properties with antibodies having different sequences or structure of the Fc domain.

Control antibody 5: The anti-FelD1 monoclonal antibody binds feline antigen and does not cross-react with human CD20 or CD3. This non-specific control IgG1 antibody was generated by the methods described in PCT Publication No. WO2013/166236 published November 7, 2013.

Control antibody 6: The anti-FelD1 antigen binding domain described in PCT Publication No. WO2013/166236 was designed as described herein to contain a chimeric IgG4 Fc domain similar to Ab1. Control Ab6 does not cross-react with human CD20 or CD3, but has a similar effector function to Ab1.

Example 3 Construction of a Chimeric Heavy Chain

Creation of chimeric heavy chains, eg chimeric CH IgG4 (SEQ ID NO: 26) and chimeric CH IgG1 (SEQ ID NO: 30), was performed using standard cloning techniques. First, a chimeric CH IgG4 was created using a two-step PCR amplification process. Two PCR fragments, fragment 1 and 2, were amplified using the starting vector construct pR001 containing wild-type hIgG4 CH DNA using primer pairs flanking the CH region, P1-P2 and P3-P4, respectively. See table. 9 below. Primers were introduced into the fragments with both the desired IgG2 lower hinge sequence (which encodes for SEQ ID NO: 52) and the flanking restriction sites. The two fragments were then joined using PCR primers P2 and P4. The resulting sequence was inserted into pR001 using Xho1-Not1 restriction sites, creating a pR002 vector construct that contains an IgG4 chimeric CH having IgG2 lower hinge sequences. The sequence was confirmed using primers P10 and P11.

In addition, chimeric CH IgG1 was created using multi-step PCR amplification. Fragment 1a was generated using primers P2 and P5 (see Table 9 below) from template pR85503 (which contains wild type human CH IgG1 DNA). Fragment 2a was amplified with primers P6 and P8 using pR002 (containing chimeric CH IgG4 DNA) as template. Fragment 3 was created using primers P7 and P9 from template pR003 (WT CH hIgG1 DNA; SEQ ID NO: 45). Fragments 1a and 2a were joined using primers P2 and P8 resulting in fragment 4. Linking fragments 2a and 3 using primers P6 and P9 created fragment 5. Fragments 4 and 5 were then fused using primers P2 and P9. The resulting sequence was inserted into pR001 using Xho1-Not1 restriction sites, creating construct pR004, which contains an IgG1 constant region with an IgG2 lower hinge region and an IgG4 CH2 domain. The sequence was confirmed using primers P10 and P11.

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Table 9. Primers for PCR generation of chimeric nucleotide constructs C _n

Primer name Primer sequence (SEQ ID NO) P1 5'- TTCGCGCAGCTTAGGTTTATGCCAGGGGGGACGGGTGGCACGGGTCGTGGTGG ACACCGT -3' (antisense) (SEQ ID NO: 63) R2 5'-AAGCTTATACTCGAGCTCTAGATTGGGAACCCGGGTCTCT-3' (SEQ ID NO: 64) RZ 5'- CCCACCGTGCCCAGCACCACCTGTGGCAGGACCATCAGTCTTCCTGTTCCCCC CAAAA-3' (SEQ ID NO: 65) R4 5'-TGTGTCTTCAGGGAGAGGGACAGAGACCCATTTACTCGCC GGCG-3' (antisense) (SEQ ID NO: 66) P5 5'- CTCGGGTTTAGAACACTGTTTTGAGTGTGTACGGGTGGCACGGGTCGTGGTGG ACACCGT-3' (antisense) (SEQ ID NO:67) R6 5'- aaatcttgtgacaaaactcacacatgcccaccgtgcccagcaccacctgtg- 3' (SEQ ID NO: 68) R7 5'- GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACAC C-3' (SEQ ID NO: 69) R8 5'- CTCTTTTGGTAGAGGTTTCGGTTTCCCGTCGGGGCTCTTG GTGTCCACATGTGG-3' (antisense) (SEQ ID NO: 70) P9 5'-CTTCAGGGAGAGGGACAGAGGCCCATTTACTCGCCGGCG-3' (antisense) (SEQ ID NO: 71) Ryu 5'-GCTGACAGACTAACAGACTG-3' (SEQ ID NO: 72) R11 5'-ATACATTATACGAAGTTATACCGGTA-3' (SEQ ID NO: 73)

Example 4 CD20xCD3 Bispecific Antibodies Selectively Bind Jurkat, Raji Cells, and Monkey T Cells Compared to Monospecific Antibodies

CD3xCD20-dtRc antibody (Control antibody 4) was compared with the monospecific Control antibodies shown in Example 2 by FACS binding assay for their ability to bind to Jurkat (CD3+, CD20 - human T cell line), Raji (CD3-, CD20+ - line human B cells) or cynomolgus macaque PBMCs (mkT cells).

FACS data were obtained using the following protocol: Cells at 2x10 ⁵ per well were incubated with antibody serial dilutions for 30 min on ice. After incubation, the cells were washed and the appropriate secondary antibody (Jurkat cells, RAJI) or secondary antibody cocktail (cynomolgus PBMC) was added and incubated for an additional 30 min. After incubation, cells were washed, resuspended in chilled PBS containing 1% BSA, and analyzed by flow cytometry on a BD FACS Canto II instrument. Jurkat and Raji cells were ranked by side and forward scatter, while cynomolgus monkey T cells were also gated in the CD2+CD4+ population. EC ₅₀ for cell binding at various titers was determined using Prism software and values were calculated using 4-parameter non-linear regression analysis. The results are shown in table. ten.

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Table 10. EC50 binding values (molar) for monospecific and _CD3xCD20 ______________ bispecific antibodies _____________

Antibody FACS-Jurkat FACS-RAJI FACS - mkT cells Control (anti-POPs) one 1.96E-10 OS OS Control (anti-POPs) 2 (+) OS 7,OSE-11 Control (anti-CO2 0) 3 Absence binding (+) OS Control 4 (AntiCD3xCD20 wild type) CH 3.85Е-08 5.99E-08 8.74Е-06

( ₊ ) EC50 values not determined, but binding observed;

OS - no binding;

NI - not explored

As shown in Table. 10, the panel of tested antibodies showed a range of binding affinities for different cell lines depending on their specificity. Bispecific Control Antibody 4 demonstrated the ability to bind to both human and cynomolgus monkey target lines. Control Antibody 4 contains the same anti-CD3xCD20 variable regions as Antibody 1 and Antibody 2 of the invention, but has a wild-type IgG1 CH region. Ahtu-CD3 Control 1 (OCT3), anti-CD3 Control 2 (SP34), and anti-CD20 Control 3 bind to Jurkat cells, cynomolgus T cells, and RAJI cells, respectively.

Example 5 CD20xCD3 Wild-type CH Bispecific Antibodies Induce Cytotoxicity Against Raji Cells in the Presence of Activated T Cells

The ability of CD20xCD3 bispecific antibodies to redirect T cell-mediated killing to CD20-expressing Raji cells was examined in an in vitro cytotoxicity assay. In addition, the ability of bispecific and parental anti-CD3 antibodies to kill U937 cells via Fc/FcR interactions was also investigated.

Kill assay with calcein was performed using the following protocol: Human and cynomolgus PBMC were isolated in a ficoll-paque gradient or with Lympholyte Mammal cell separation medium, respectively. Isolated PBMCs were activated for several days in medium containing recombinant human IL-2 (30 U/ml) and T-cell activation beads (anti-CD3/CD28 for human PBMC, anti-CD2/CD3/CD28 for cynomolgus PBMC).

Target cells (Raji for CD20-mediated killing and U937 for FcR-mediated killing) were labeled with calcein and incubated with activated T cells at an effector:target ratio of 10:1 using 3-fold serial antibody dilutions for 3 h at 37 °C After incubation, the plates were centrifuged and the supernatants were transferred to a translucent black plate with a transparent bottom for fluorescence analysis. EC ₅₀ , that is, the molar concentration of the bispecific antibody that induces 50% cytotoxicity, was determined using Prism. Values were calculated using 4-parameter non-linear regression analysis. The results are summarized in table. ten.

Table 11. EC ₅₀ values for CD20 x CD3-induced cytotoxicity against ____________________ Raji and U937 cells _______________

Antibody Cytotoxicity of Raji, human T cells [M] Cytotoxicity of Raji, monkey T-cells [M] Cytotoxicity U937, Human T cells [M] Control Ab 1 (anti-POPs) OA OA 3.04Е-12 Control AB 4 (AntiCD3xCD20) 5.63E-11* 1.27E-12* 8.86Е-11*

(*) Data are median values from 3 or more independent experiments.

Data without (*) are representative/mean values from 1 or 2 independent analyses.

OA=No Activity

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As shown in Table. 11, a CD20 x CD3 bispecific antibody containing human-specific and cynomolgus cross-reactive anti-CDS arms and a wild-type IgGl CH region was able to specifically redirect cytotoxicity to Raji cells in the presence of activated human T cells. In the presence of activated cynomolgus monkey T cells, killing of Raji occurred when they were incubated with the control AL4 bispecific antibody, which has an anti-CDS arm that activates monkey T cells. Both the specific antibody and the control AI (anti-CDS mAb) showed activity in the U937 Fc/FcR dependent kill assay. This activity can be blocked by adding blocking non-specific human IgG to the reaction (Data not shown).

Example 6 CD20 x CD3 Bispecific Antibodies Containing Chimeric CH Regions Demonstrate Reduced Effector Function in a CDC Assay

The CD20xCD3 bispecific antibodies with chimeric CH regions (Antibody 1 and Antibody 2) described in Example 2 were designed to alter or reduce their effector function. Compared to antibodies containing the heavy chain constant region of the wild-type (wt) IgGl isotype (Control AL4), amino acid substitutions in the CH region may interfere with the ability of Fc Ig to bind to its receptor(s). Consequently, signaling and immune responses such as B cell activation or phagocytosis may be altered. The effect of amino acid modifications in the CH region on the effector function of complement-dependent cytotoxicity (CDC) (in this example) and antibody-dependent cell-mediated cytotoxicity (ADCC) (see Example 7) was investigated.

To investigate the effect of Antibody 1 and Antibody 2 on CSC effector function, CD20 expressing Raji (target) cells (5000/well) or Daudi cells were seeded in the presence of 5% human serum complement. Serial dilutions of Antibody 1, Antibody 2 and control antibodies, starting at 100 nM, were added to the cells for 4 hours at 37°C. Target cell lysis was determined using the CytoTox Gio™ kit (Promega) and the percentage of cytotoxicity was calculated.

The percentage of cytotoxicity was calculated using the equation:

% cytotoxicity= ( (L _s -L _sr ) / (L _mr -L _sr ) ) *100%, where L _S r is the base luminescence of target cells and L _MR is the maximum release of calcein from cells lysed with digitonin. EC ₅₀ for cytotoxicity was determined using Prism software (GraphPad). The values calculated using 4-parameter non-linear regression analysis are given in Table. 12 and in FIG. 5A and 5B.

The CSC activity of Antibody 1 and Antibody 2 against Daudi and Raji cells is significantly reduced compared to the corresponding antibody having a heavy chain constant region of dt. See table. 12 and FIG. 5A/B. Some CDC activity was observed for Antibody 1 against Raji cells, however, the overall results show that the chimeric antibodies elicit weaker effector responses than the control IgGl antibodies with Fc gt.

Table 12. CD20xCD3 Bispecific Antibodies Containing Chimeric CH Regions Show Decreased Activity in the CSC Assay to Determine Effector Function

KZTs Target Cell^ Daudi Raj i _EC50 [M] Maximum cytotoxicity (%) _EC50 [M] Maximum cytotoxicity (%) Control TMe 4 6.12E-08 ~95 1.98E- 08 ~85 Ido 1 OA OA 2.8 6E- 08 ~45 imo 2 OA OA 3.49E- 08 ~10

About A: Lack of activity

Example 7 CD3xCD20 Bispecific Antibodies Containing Chimeric CH Regions Demonstrate Reduced Effector Function in ADCC Assay

To investigate the effect of Antibody 1 and Antibody 2 versus a bispecific antibody with wild-type CH regions (and identical variable regions) on ADCC effector function, freshly isolated unstimulated CD56-positive NK cells or E1K92 cells engineered to express higher affinity V -allele FcyRIIIa, were seeded with calcein-labeled CD20-positive Raji or Daudi cells in the presence of antibodies with chimeric CH (Antibody 1 and Antibody 2) and a control antibody with CH dt (Control antibody 4). The release of calcein from target cells was monitored and the percentage of cytotoxicity was determined. Percent cytotoxicity and EC50 were calculated as described for the CSC analysis above. The results are shown in table.

-46039429 and in Fig. 6A and 6B.

Chimeric CH antibodies, Antibody 1 and Antibody 2, do not mediate ADCC activity (Table 13) against Raji or Daudi cells.

Table 13. CD3 x CD20 bispecific antibodies containing chimeric CH regions show reduced activity in the ADCC assay to determine effector function

ADCC Target cells -> Daudi Raj i EC50 [M] Maximum cytotoxicity (%) EU50 [M] Maximum cytotoxicity (%) Control AB 4 1.87E-10 ^70# 1.48Е-09 -65 ^# Ab 1 OA OA OA OA AB 2 OA OA OA OA

OA: Lack of activity;

#: background cytotoxicity ~20%

Example 8 Binding Affinities and Kinetic Constants of Chimeric Antibodies Obtained by Surface Plasmon Resonance

Ahth-CD3 x anti-CO20 bispecific antibodies having chimeric heavy chain constant regions, Antibody 1 and Antibody 4, were analyzed by surface plasmon resonance (SPR) (Biacore) to determine their binding kinetic parameters to human and cynomolgus monkey Fcy receptors. Isotype controls, i.e. gt-IgGl isotype control and CPPC isotype control gt-IgGd, were tested by the same method.

Briefly, SPR experiments were performed at 25° C. on a Biacore T200 instrument using a carboxymethyl dextran coated chip (CM-5). A mouse anti-pentahistidine monoclonal antibody (GE Healthcare) was immobilized on the surface of a CM-5 sensor chip using standard amine conjugation. 140 RU - 376 RU His-tagged human or simian FcyR proteins were captured on an amine-coupled immobilized anti-pentahistidine antibody on a CM-5 chip, and antibody stock solutions were injected over the captured proteins at 20 µl/min for 2.5 min. The binding reaction of nAb was monitored and steady state binding was calculated for low affinity receptors. The kinetic rate constants of association (k _a ) and dissociation (k _d ) were determined by processing and fitting the data with a 1:1 binding model using Scrubber 2.0 curve fitting software. Equilibrium dissociation constants (Kd) for binding and dissociative half-life (ti _/2 ) were calculated from kinetic rate constants as: Kd (M)=k _d /k _a ; and ti _{/ 2} (min) \u003d (1p2 / (60 * k _d ).

Table 14 Binding Kinetic Parameters for Wild-Type (wt) Heavy Chain and Chimeric Heavy Chain Antibodies

Binding to His-captured human FcyRI at 25°C Antibody k _a (M ^-1 with H k" Cs- ¹ ) Kd ( ^10-9 M) T ^1/2 (min) Isotype control dt-IgGl 1.74E+05 7.48Е-04 4.3 fifteen Isotype control flT-IgG4 СРРС 1.71E+05 2.36E-03 13.9 5 Ab 1 OS OS OS OS AB 2 OS OS OS OS

OS: No Detectable Binding

As the results in Table. 14, the bispecific antibodies, Antibody 1 and Antibody 2, show no binding to human FcyR! compared to antibodies having a wild-type (wt) hlgGl or h!gG4-CPPC CH region in a SPR assay. The chimeric heavy chain bispecific antibodies of the present invention also show weak or no binding to several low affinity human FcRy receptors (e.g., weak binding to human FcRyllA, FcRyllB, and no detectable binding to human FcRyl, FcRylllA or FcRylllB) compared to antibodies with Fc-sequence dt hlgGl or

-47039429 hIgG4-CPPC (Table 15 below).

Table 15. Steady state binding for heavy chain antibodies

Example 9 Pharmacokinetic Profile of Chimeric Antibodies

The toxicokinetic profile of Antibody 1 and Antibody 2 was assessed by obtaining blood samples from male cynomolgus monkeys (3 animals/treatment group) receiving one 30 minute BB infusion followed by a 12 week observation period. Blood samples for toxicokinetic analysis of total drug concentration in the blood serum were taken before dosing and after dosing at 5 minutes and 5, 24, 48, 72 and 168 hours, and at 14, 21, 35, 49, 66 and 84 days. The obtained serum samples were analyzed by direct enzyme immunosorbent assay (ELISA) to determine the total concentration of the drug Ab 1 or Ab 2. The toxicokinetics of the study drugs were evaluated using a non-compartmental analysis (Phoenix WinNonLin) to determine the pharmacokinetic parameters. The results are shown in table. 16 (AUC = area under the concentration-time curve; C _max = observed maximum serum concentration).

Table 16. Pharmacokinetic profile of chimeric antibodies in cynomolgus monkey serum after a single intravenous infusion

Parameter Units 1 mg/kg AB 2 1 mg/kg AB 1 Average SO Average SO Smake mcg/ml 33.4 3.79 26.0 4.72 Cmax/dose kg*mcg/ml/mg 33.4 3.79 26.0 4.72 Tmax day 0.0243 0 0.0243 0 PPKo-168 h day*mcg/ml 100 20, 1 61.1 8.04 PPCo-168 h / dose day*kg*mcg/ml/ mg 100 20, 1 61.1 8.04 Т1/2 Day 8.14 1, 15 14.0 2, 64

After a single intravenous dose of 1.0 mg/kg Ab 1 and Ab 2 in cynomolgus monkeys, mean peak concentrations (C _max ) of 33.4 and 26.0 µg/ml, respectively, and mean AUC values of _0-168h 100 and 61.1 day*mcg/ml, respectively. The apparent terminal half-life was estimated to be 8.14-14.0 days for these two molecules. The data indicate that continuous exposure to Ab 1 and Ab 2 was maintained in all animals for most of the 12-week observation period, with exposure being comparable across all treatment groups. No apparent immunogenicity of the studied preparations was observed. The general pharmacokinetic profiles of Ab 1 and Ab 2 are typical of monoclonal antibodies administered to cynomolgus monkeys.

Example 10 CD3 x CD20 bispecific antibodies can reduce CD20+ B cells in cynomolgus monkeys at lower doses than monospecific antibody

To determine the in vivo efficacy of administration of the CD3xCD20 bispecific antibody, Antibody 1 and Control Antibody 4, changes in peripheral blood levels of CD20+ B cells in cynomolgus monkeys were examined by FACS after administration of an anti-CD3xCD20 bispecific antibody compared to a monospecific anti-CD20 antibody (Control Ab 3). The study was performed on male cynomolgus monkeys (Macaca fascicularis) divided into eight dosing groups of 3 animals per dosing group as follows: Group 1 was the placebo group (stock solution administration); Group 2 received a monospecific antibody (Control Ab 3; ri- 48 039429 tuxane) at a dose of 30 mg/kg (30 mg/kg in monkey is equivalent to a human dose of 375 mg/m ² which is considered the maximum clinical dose); Group 3 received the bispecific CD3xCD20 Control antibody 4 at a dose of 0.01 mg/kg; Group 4 - Antibody 4 at a dose of 0.1 mg/kg; Group 5 - Antibody 4 at a dose of 1 mg/kg; Group 6 - Antibody 1 at a dose of 0.01 mg/kg; Group 7 - Antibody 1 at a dose of 0.1 mg/kg; and Group 8 - Antibody 1 at 1 mg/kg. Blood was taken at day -7 and day -4 before dosing the animals. Doses of antibody preparation or stock solution (placebo) were administered by iv. infusion, and blood was taken on days 2, 4 and 7 after dosing. Blood samples were analyzed by FACS for markers of B cells (CD20; Table 17) and T cells (CD3, see below) and the absolute number of these cell types was determined.

Briefly, 100 µl of blood was incubated for 3 minutes with 1.5 ml of CCT lysis buffer in Eppendorf tubes. Cells were centrifuged for 5 min at 0.4xg, washed 2x with FACS wash buffer (PBS + 3% PBS) and blocked for 10 min at room temperature with Fc blocking reagent. The cells were then incubated for 30 min at 4°C with directly conjugated antibodies to hCD45 and CD20 with fluorescent reagents. B-cell (CD20) or T-cell (CD3) subgroup quantitation was first performed using a heterogeneous gating strategy consisting of CD45 fluorescent staining and side scatter characteristic (SSC) demarcation (CD45brightSSCdim) to establish white blood cell (BCT) populations. . B cell populations were then determined by using relevant fluorescently labeled antibodies (CD20 APC-Cy7). After staining, cells were washed twice before FACS evaluation with a FACSCanto cytometer and analysis with FlowJo software. The results are shown in table. 17 and in FIG. 11A and 11B.

Table 17. Number of circulating CD45, CD20-positive cells in monkey ____________ peripheral blood after ____________ treatment

CD20+ cells (U/µl) on the day of the study Treatment Animal No. -7 -four 2 four 7 78 1.87 2.69 1.85 2.09 1, 62 placebo 79 1.28 1.31 0.98 1.24 0.98 80 2.41 2.90 2.23 2.71 1.78 Control /Until 3 81 0.71 0.80 0.00 0.00 0.00 82 0.97 2.49 0.00 0.00 0.00 83 0.71 1.28 0.00 0.00 0.00 CD3xCD20-dtx 84 2.00 2.82 0.03 0.02 0.03 (AL 4) 85 1.23 1.96 0.00 0.00 0.00 0.01 mg/kg 88 1.50 2.29 0.01 0.00 0.00 CD3xCD20-dtx 87 0.79 1.20 0.00 0.00 0.00 (AL 4) 88 1.72 3.05 0.00 0.00 0.00 0.1 mg/kg 89 0.28 0.60 0.00 0.00 0.00 CD3xCD20-dtx 90 0.63 1.02 0.00 0.00 0.00 (AL 4) 91 0.66 0.65 0.00 0.00 0.00 1 mg/kg 92 0.56 1.50 0.00 0.00 0.00 AB 1 0.01 mg/kg 93 94 95 1.16 0.72 1.95 1.96 1.49 1.94 0.00 0.00 0.02 0.00 0.04 0.02 0.00 0.00 0.01 AB 1 0.1 mg/kg 96 97 0.48 1.30 0.60 1, 82 0.00 0.00 0.00 0.00 0.00 0.00 98 4.87 5.00 0.00 0.00 0.00 AB 1 99 0.23 0.34 0.00 0.00 0.00 1 mg/kg 00 1.39 1.93 0.00 0.00 0.00 01 2.29 2.30 0.00 0.00 0.00

As shown in Table. 17 and in FIG. 11A, administration of the CD3xCD20 bispecific antibody and the anti-CD20 monospecific antibody resulted in a decrease in the number of circulating B cells to baseline at the first time point measured (day 2). This reduction was not observed in the placebo control animals. The reduced number of B cells in the bispecific groups persisted for 1 week after the 1 mg/kg dosing of the bispecific antibodies, and also the reduced number of B cells persisted in the bispecific groups with the 0.01 and 0.10 mg/kg dose.

Also in this experiment, T-cell (CD3+) levels were monitored with fluorescently labeled aHTu-CD3 antibodies. A transient decrease in the number of circulating T cells was observed on day 2 post-dosing in the bispecific antibody groups (Ab 4 and Ab 1; all doses). The stock solution control group (placebo) and the Ab 3 control group (rituxan) did not show a decrease in the number of T cells (below baseline) at the time points measured. T cell levels recovered to baseline in the bispecific antibody groups on day 4 and remained at baseline until the end of the experiment (see Figure 11B).

In vivo potency of the CD3xCD20 bispecific antibodies, Antibody 1 and Antibody 4, was determined in the peripheral blood of cynomolgus monkeys in a long-term (3 months) study measuring changes in CD20+ B cell levels or CD3+ T cell levels, similar to the assay described above. Placebo (stock solution) or bispecific antibodies were administered at a dose of 1.0 mg/kg on day 0. Peripheral blood B cell levels were significantly reduced on day 2 in both bispecific antibody groups and remained reduced throughout the study in all samples except placebo (see Fig. 12A). A transient decrease in T cell counts was observed on day 2 in the bispecific groups, then T cell counts recovered to baseline levels on day 4 and remained within baseline as measured throughout the study (>80 days) for animals treated with bispecific antibodies ( Fig. 12B). Placebo-treated animals did not show a transient decrease in T-cell numbers.

To further determine the in vivo efficacy of the CD3xCD20 bispecific antibodies, Antibody 1 and Antibody 4, at low doses, a long-term (2 month) study in peripheral blood of cynomolgus monkeys measured changes in CD20+ B-cell levels or CD3+ T-cell levels, similar to the experiments described above. Bispecific antibodies were administered at 0.01 mg/kg or 0.001 mg/kg (1 µg/kg) on day 0. Peripheral B-cell levels were significantly reduced on day 2 and remained reduced throughout the study in both CD3xCD20 groups (FIG. 13A), similar to what was observed for animals treated with higher doses of CD3xCD20 bispecific antibodies (as seen in Table 17 and FIG. 11A or 12A). Animals treated with very low doses (1 μg/kg) of the bispecific antibodies showed the same transient decrease in T cell numbers and recovery as was observed in animals treated with higher doses of CD3xCD20 bispecific antibodies (see Figure 13B vs. Figure 11B or 12B).

The decrease in the number of B cells observed in the described studies correlated with the decrease in the number of circulating antibodies in the peripheral blood of animals treated with CD3xCD20-chimeric Fc Antibody 1 (see Fig. 14). As the amount of antibody in the circulation of the animals decreased over time, B cell populations began to recover (eg, as observed at day 81 for animal #I06881).

A correlation of a decrease in the number of B cells with a decrease in the amount of circulating antibody in the peripheral blood was also observed for animals treated with CD3xCD20-chimeric Fc Antibody 2 (see Fig. 15), given that when the amount of antibody in the animal circulation decreased over time, populations of B- cells began to recover, for example, as observed at day 66 for animal # I06876 and at day 68 for animal # I06877. A similar correlation was also observed for animals treated with the CD3xCD20-dtFc bispecific antibody (Ab 4) (see FIG. 16). As the amount of antibody in the circulation of the animals decreased over time, B cell populations began to recover (see FIG. 16, for example, as observed at day 91 for animal #I06870 and at day 64 for animal #I06872).

Example 11 CD3xCD20 bispecific antibodies can reduce the number of CD20+ B cells in cynomolgus monkey lymphoid tissues at lower doses than monospecific antibody

Changes in CD20+ B cell levels in cynomolgus monkey lymphoid tissues were examined by FACS after administration of an anti-CD3xCD20 bispecific antibody (Antibody 1 or Antibody 4) versus a monospecific anti-CD20 antibody (Control Ab 3 - Rituxan). The study was performed on male cynomolgus monkeys (Macaca fascicularis) divided into eight dosing groups of 3 animals per dosing group, similar to Groups 1-8 of Example 10 above. Doses of the antibody preparation or stock solution were administered by i.v. infusion, and the animals were sacrificed and received tissues on the 7th day after dosing. Tissue samples were analyzed by FACS for white blood cells (CD45+) and in particular B cell markers (CD20+), then the % B cell volume was determined.

B cell populations were determined using relevant fluorescently labeled antibodies (CD20 APC-Cy7) and FACS assessment, similar to the method described above in example 10. The results are shown in table. 18 and in FIG. 17A and 17B.

- 50 039429

Table 18 Percentage of CD20-positive cells in simian mesenteric lymph nodes and spleen after

Mesenteric lymph nodes Spleen Treatment Animal No. Day 7 Day 7 placebo 78 38.14 63.22 79 38.57 62.79 80 37.36 49, 17 Control AB 3 81 6.21 4.5 82 10.3 3.45 83 4.21 2.18 AB 4 0.01 mg/kg 84 13.43 3, 14 85 6, 88 2.27 86 10.78 1.39 AB 4 0.1 mg/kg 87 1.51 2.37 88 0.45 1.65 89 1.24 2.4 AB 4 1 mg/kg 90 0.63 0.97 91 0.62 1.93 92 1, 08 1.22 AB 1 0.01 mg/kg 93 5, 38 1.22 94 6, 37 1.89 95 13.25 6.99 AB 1 0.1 mg/kg 96 0.43 1.55 97 0.68 1.75 98 2.36 2.97 AB 1 1 mg/kg 99 0.33 1.79 00 16 1.71 01 0.5 1.21

As shown in Table. 18 and in FIG. 17A, administration of CD3xCD20 bispecific antibodies compared with anti-CD20 monospecific antibody resulted in a decrease in the number of tissue B cells in the spleen at lower doses (0.01 to 1.0 mg/kg doses) for the bispecific groups. This reduction was not observed in the placebo control animals.

As shown in Table. 18 and in FIG. 17B, administration of CD3xCD20 bispecific antibodies compared with anti-CD20 monospecific antibody resulted in a decrease in the number of tissue B cells in mesenteric lymph nodes at lower doses (0.01 to 1.0 mg/kg doses) for the bispecific groups, while the bispecific antibody dose of 0.1 mg/kg and 1 mg/kg resulted in a more effective reduction in the number of B cells than in the monospecific group. This reduction was not observed in the placebo control animals.

Example 12 Treatment of CD3xCD20 tumors with a bispecific antibody

A. CD20xCD3 Bispecific Antibody Treatment Suppresses the Growth of Raji Tumors in NSG Mice

To evaluate the efficacy of selected anti-CD3xCD20 bispecific antibodies in reducing the growth of Raji tumors, NSG mice (NOD/LtSz-scid/IL2Rγ ^null mice) purchased from Jackson Laboratories (Bar Harbor, Maine, USA) were implanted subcutaneously with 2x10 ⁶ Raji tumor cells. and 5x105 human PBMCs (day 0). On the same day, mice were treated with an intraperitoneal dose of 0.4, 0.04, or 0.004 mg/kg per mouse (N=5 mice per treatment group) of Antibody 1 or Control Antibody 5 (IgG1 antibody to an irrelevant target), or stock solution. Beginning on day 0, mice were treated twice a week with an intraperitoneal dose of the drug or stock solution for the remainder of the study. Tumor volume was measured twice a week using calipers and tumor volume was calculated as: Volume=(length x width ² )/2. Statistical analysis was performed using GraphPad Prism 5.0 software (Macintosh version).

Statistical significance was determined using two-way analysis of variance

- 51 039429

ANOVA with Tukey's post hoc test of multiple comparisons. Data for each indication were compared across treatment groups. The p<0.05 threshold was considered statistically significant.

The results are illustrated in FIG. 7A-7F. These results show that Antibody 1 (CD3xCD20 chimeric Fc) targets Raji tumors in mice also implanted with human immune cells and results in complete suppression of tumor growth at the doses studied (Fig. 7C: 0.4 mg/kg Ab1; Fig. 7E: 0.04 mg/kg Ab1, Fig. 7F: 0.004 mg/kg Ab1). This example demonstrates that treatment of CD3xCD20 with bespecifically Antibody 1 was effective in inhibiting tumor growth from the time tumors were implanted. The growth of Raji tumors was completely suppressed up to 23 days after implantation in mice that received doses of 0.4, 0.04 or 0.004 mg/kg of Antibody 1 compared to controls. It was also observed that neither Antibody 1 nor Control antibody had a significant effect on the body weight of mice during the study (data not shown).

The antitumor activity of the CD3xCD20 bispecific antibodies was further investigated in a similar NSG mouse model (described above), with NSG mice dosed with 1 mg mouse IgG (mIgG2a Fc) on day -1 and once a week thereafter. The results are illustrated in FIG. 8A-8B.

This experiment demonstrates that treatment of CD3xCD20 with Bispecific Antibody 1 (CD3xCD20-chimeric Fc) or CD3xCD20 with Bispecific Antibody 4 (CD3xCD20-dtFc) was effective in inhibiting tumor growth from the time of tumor implantation, at doses of the bispecific Ab tested in the presence of circulating IgG. (FIGS. 8A-8B). As seen in FIG. 8A, Antibody 1 (CD3xCD20 chimeric Fc bispecific antibody) demonstrates complete suppression of tumor growth during the study period with or without IgG supplementation in this experiment.

B. Treatment of CD20xCD3 with a bispecific antibody reduces established tumors in NSG mice

The efficacy of selected anti-CD3xCD20 bispecific antibodies in reducing established tumors in NSG mice was evaluated. NSG mice (NOD/LtSz-scid/IL2Ry ^nul ' mice) were purchased from Jackson Laboratories (Bar Harbor, Maine, USA) and subcutaneously implanted with HLA syngeneic Raji tumor cells (2x10 ⁶ ) and human PBMCs (5x105) (- day 15). Before treatment, the tumors were allowed to develop in the host for 15 days. The day before drug administration (-1 day), each mouse was dosed with an additional 5 mg Fc mIgG2a. Thereafter, mice were dosed with 5 mg Fc mIgG2a per mouse once a week for the duration of the experiment (7, 14 days, etc.). Prior to drug administration, mice were divided into 2 experimental groups according to tumor size: Group 1: ~200-400 mm ³ or Group 2: ~500-900 mm ³ .

Following drug treatment, each mouse was monitored and recorded for tumor size at days 0, 3, 6, 10, 14, 17, 21 and 28. Tumor size was measured twice a week using calipers and tumor volume was calculated as: Volume=(length x width ² )/2. The presence of tumors was also determined by palpation. Statistical analysis was performed using GraphPad Prism 5.0 software (Macintosh version). Data for each indication were compared across treatment groups. The results are illustrated in FIG. 9 and 10.

a) Group 1: ~200-400 mm ³ tumors. Starting from day 0, several groups of 4 or 5 mice each were treated with the indicated dose of the drug or stock solution once a week (i.e. on days 7, 14, etc.) as follows:

i) Control: stock solution only ii) Control antibody 5, 0.4 mg/kg iii) Antibody 1 (CD20xCD3 chimeric Fc), 0.4 mg/kg

b) Group 2: ~500-900 mm ³ tumors. Starting from day 0, several groups of 4 mice each were treated with the indicated dose of the drug once a week (i.e. on days 7, 14, etc.) as follows:

i) Control antibody 5, 0.4 mg/kg ii) Antibody 1 (CD20xCD3 chimeric Fc), 0.4 mg/kg

Tumors completely regressed in the 0.4 mg/kg Antibody 1 (CD20xCD3 chimeric Fc) group on day 14 (See FIG. 9).

In a model with larger advanced tumors (i.e. Group 2, ~500-900 mm ³ ), treatment with Antibody 1 (CD20xCD3 chimeric Fc) (0.4 mg/kg) resulted in complete ablation of advanced tumors in mice at 21 day (See Fig. 10).

Example 13 CD3xCD20 bispecific antibodies induce PBMC proliferation in vitro

The ability of selected CD3xCD20 bispecific antibodies and control constructs to stimulate and induce proliferation of peripheral blood mononuclear cells (PBMCs) was assessed by ATP-catalyzed quantification (CellTiter Glo®). Activation of PBMCs results in the release of cytokines that trigger cell proliferation.

Proliferation data were obtained using the following protocol: Human or cynomolgus monkey-derived PBMCs (5x105/well) were incubated with serial dilutions of CD3xCD20 bispecific antibody or control antibody and commercial anti-CD28 antibody (human: 200 ng/mL, cynomolgus monkey: 500 ng /ml) for 72 h at 37°C. Cells were quantified with a Cell Titer Glo® and luminescence, as an indicator of cell viability, was measured with a VICTOR X5 multi-label plate reader (PerkinElmer) to detect live cells. Cell viability (induction fold for stimulated versus unstimulated cells) was determined by dividing stimulated cell luminescence by baseline unstimulated cell luminescence and plotted using Prism software. The results are summarized in table. 19 and in FIG. 18A and 18B.

Table 19. EC ₅₀ for human and cynomolgus PBMC proliferation induced by anti-CD3xCD20 bispecific antibodies

Data are median values from 3 or more independent analyses.

OA=no activity.

As shown in Table. 19 and in FIG. 18A-18B, both CD20xCD3 bispecific antibodies of the invention induced human and cynomolgus PBMC proliferation. Antibody 1 induced human and cynomolgus PBMC proliferation with approximately the same efficiency. Control Ab 5 showed no activity.

Example 14 CD20 x CD3 bispecific antibodies mediate cell killing by activated T cells in vitro

Human or cynomolgus PBMCs were isolated in a Ficoll-Pak gradient or with Lympholyte Mammal separation medium, respectively. Isolated PBMCs (1 x 10 ⁶ cells/ml human PBMC or 5 x 10 ⁶ cells/ml cynomolgus monkey PBMC) were activated for 7 and 21 days, respectively, in complete medium (RPMI supplemented with 10% PBS, 100 U/ml penicillin, 100 μg/ml). ml streptomycin, 292 μg/ml L-glutamine) containing recombinant human IL-2 (30 U/ml for human PBMC, 100 U/ml for cynomolgus PBMC) and T-cell activating beads (anti-CD3/CD28 for PBMC human, anti-CD2/CD3/CD28 for cynomolgus monkey PBMC). CD20-expressing Raji cells (2×10 ⁶ cells/ml) were labeled with 8 μM calcein-AM for 30 min at 37° C. and washed three times with medium. Calcein-labeled target cells (10,000-20,000 cells/well) were plated in 200 µl of activated T cells (effector:target ratio 10:1) and serial dilutions of Antibody 1, Ab 4, or Control Ab 5 (human concentration range: 2 to 0.00003 nM; concentration range for cynomolgus monkey: 6.6 nM to 0.002 pM) in complete medium for 2 hours at 37°C. After incubation, the plates were centrifuged and the supernatants were transferred to a translucent black plate with a transparent bottom for fluorescence analysis. Percent cytotoxicity was calculated using the equation:

% cytotoxicity=((FS - FSR)/(FMR-FSR))*100%, where FS is the release of calcein from the test well, FSR is the spontaneous release of calcein, and FMR is the maximum release of calcein from cells lysed with Triton-X.

Cell viability EC ₅₀ (ATP catalyzed quantification) was determined using Prism software. Cell lysis (cytotoxicity) was assessed by the release of calcein as a fraction of the maximum release. The percentage of cellular cytotoxicity was calculated as observed release versus maximum release and EC ₅₀ values were determined. The results are shown in table. 20 and in FIG. 19A (human T cells) and 19B (monkey T cells).

Table 20. EC ₅₀ values for CD3xCD20-induced cytotoxicity in _________________ against Raji cells_____________________

Antibody Control AB 5 Cytotoxicity Raj i, activated human T cells [M] OA Raji Cytotoxicity, Activated Monkey Cells [M] NO CD3xCD20-dtes (AL 4) 1.571E-11 1,730E-12 Antibody 1 2.503E-11 9.104E-12

OA=No activity;

NI=not explored.

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As shown in Table. 20, Antibody 1 mediated targeted cell killing with typical EC5o values of 25.0 and 9.10 pM for human (FIG. 19A) and cynomolgus monkey (FIG. 19B) T cells, respectively. Antibody 4 mediated targeted cell killing with typical EC ₅₀ values of 15.7 pM and

1.73 pM for human (Fig. 19A) and cynomolgus monkey (Fig. 19B) T cells, respectively. No activity was observed for the control antibody.

To investigate the specific killing of CD20-bearing target cells by flow cytometry, parental B16F10.9 mouse myeloma cells (which do not express CD20) and B16F10.9 cells engineered to stably express human CD20 (B16F10.9/CD20) were labeled 1 μM fluorescent dyes-witnesses-carboxyfluorescein succinimidyl ester (CFDA-SE) and Violet Cell Tracker, respectively. After labeling, the cells were mixed in a 1:1 ratio and seeded overnight at 37°C. Human PBMCs were plated separately in RPMI supplemented medium at 1x106 cells/ml and incubated overnight at 37°C to achieve lymphocyte enrichment by reducing adherent macrophages, dendritic cells and some monocytes. The next day, target cells were incubated with adherent, cell-depleted naïve PBMCs (effector:target ratio 4:1) and serial dilutions of test CD3xCD20 bispecific antibodies or Control IgG1 antibody 5 (concentration range: from 66.7 nM up to 0.25 pM) for 48 hours at 37°C. Cells were removed from cell culture plates using enzyme-free cell dissociation buffer and analyzed by FACS. For FACS analysis, cells were stained with far red cell tracker for dead/live cells (Invitrogen). To evaluate the specificity of killing, cell gating was performed on live populations labeled with Violet and CFDA-SE. The percentage of each population was used to calculate adjusted survival in the following way:

Adjusted survival=(R1/R2)*100 where

R1=[(B16F10.9/CD20)/background cells (B16F10.9)]*100 in the absence of antibody, a

R2=to the same ratio, but in the presence of the test antibody.

Table 21. EC ₅₀ values for specific target killing for B16F10.9/CD20 cells

Antibody % Cell Survival B16F10.9/CD20 [M] Control AB 5 OA CD3xCD20-dtEc (Ab 4) 1.282E-11 Antibody 1 1,952E-11

OA=No activity.

Both CD3xCD20-chimeric Fc (Antibody 1) and CD3xCD20-dtFc (Antibody 4) specifically directed human T cells to kill only CD20-expressing target cells (Fig. 20A-B) in a mixed cell population. Targeted cell killing was observed only in the presence of the bispecific antibody, with B16F10.9/CD20 cell numbers reduced in a dose-dependent manner by Antibody 1 (EC ₅₀ 19.5 pM) and Antibody 4 (EC ₅₀ 12.8 pM) (FIG. 20B). Less than 5% of CD20-expressing cells remained alive at the highest dose tested (10 μg/ml) (FIG. 20B). No evidence of cell death was observed in the B16F10.9 parental cell population or in the B16F10.9/CD20 population using Control Ab 5, a control IgG1 antibody.

Example 15 CD3xCD20 Bispecific Antibody Upregulates Early Activation Marker CD69 on T Cells in a 20 Hour In Vitro FACS Assay

CD69+ is one of the earliest inducible cell surface markers that is indicative of T cell activation. T cell activation can be determined by examining the upregulation of specific cell surface markers such as CD69.

The ability of the CD3xCD20 bispecific antibody to upregulate the early activation marker CD69 on human or cynomolgus monkey T cells in whole blood was determined using a 20 hour in vitro FACS assay. Briefly, T cell activation was assessed by incubating freshly isolated human or cynomolgus whole blood (100 µL) in flat-bottomed 96-well plates with 5-fold (human) or 10-fold (cynomolgus) serial dilutions of Antibody 1, Antibody 4, or Control Ab 5 (concentration range: 50 nM to 0.0006 nM) in RPMI+L-glutamate with a final volume of 200 μL for 20 h at 37°C. After incubation, the plates were centrifuged for 5 min at 1000 rpm and the plasma was removed. To determine the upregulation of CD69 on T cells, a phenotyping cocktail containing directly conjugated antibodies to CD2 and CD69, as well as CD45, CD4, CD8 and CD19 (human) or CD16 (cynomolgus monkey) was added directly to the blood for 30 min. at 4°C. Red blood cells were lysed for 15 min with 1.5 ml PharmLyse buffer following the manufacturer's instructions. Cells were washed twice, resuspended in 200 μL of chilled PBS + 1% PBS and analyzed by flow cytometry using a BD FACSCanto cytometer. CD4+ T cells were identified by first gating on viable CD45+ small lymphocytes and then gating on CD19-/CD2+/CD4+ T-cells (human) or CD16-/CD2+/CD4+ T-cells (cynomolgus monkey) .

Percentage of activated (CD69+) T cells in total effector cells is shown

CD2+. See table. 22 as well as FIG. 21. The results show that CD3xCD20 bispecific antibodies significantly activated T cells, as evidenced by the early activation marker CD69.

Table 22. EC ₅₀ values for specific target killing for B16F10.9/CD20 cells

Antibody % Activated T cells (CD69+) [M] Control AB 5 OA CD3xCD20-dtx (AJo 4) 7.907E-11 CD3xCD20-chimeric Fc (Antibody 1) 1,560E-11

OA=No activity.

Example 16 CD3xCD20 Bispecific Antibodies Induce T Cell Clustering with Target Cells

A cell clustering format was used to determine that a CD3xCD20 bispecific antibody bound T cells to target cells (CD20+ cells) via its binding arms. Effector cells were pre-stained with CFSE and CD20+ cells were pre-stained with Violet Cell Tracker for 24 h and gated in separate quadrants after incubation with an irrelevant control antibody (Control antibody 5, an irrelevant IgG1 isotype antibody). See fig. 22A, which demonstrates the absence of clustering (double staining) in a mixture of cells when treated with an irrelevant antibody. After incubation with the CD3xCD20 bispecific antibody, cell clusters appeared due to CFSE and Violet staining (see FIG. 22B, upper left quadrant of the scatterplot, bold square area).

Example 17 Expression of Inhibitory Markers Tim-3 and PD-1 on CD3+ Cells

T-cell dysfunction or depletion occurs in tumor-bearing host organisms. Tim-3 and PD-1 receptors have been identified as markers of T cell depletion in chronic disease states. According to Sakuishi, K. et al. (J. Exp. Med. 207 (10): 2187-2194, 2010), tumor infiltrating lymphocytes (TIL) that are positive for Tim-3 and PD-1 (Tim-3+PD-1+TIL) show the most severe wasting phenotype, defined by the absence of proliferation and production of IL-2, TNF and IFN-γ.

CD3-positive cells were isolated from the blood and tumors of NSG mice implanted subcutaneously with HLA syngeneic Raji tumor cells and human PBMCs - see Example 12B above. Briefly, tumors were allowed to develop in the host for 15 days prior to treatment, then the mice were divided into two treatment groups based on the size of the tumors (see Example 12B). Blood was obtained on day 9 from treated (bispecific Ab) and untreated mice from each study group, ie. Group 1, ~200-400 mm ³ or Group 2, ~500-900 mm ³ . Untreated mice (stock solution or Control Ab) having tumors reaching 1500 mm ³ were sacrificed at the end of the study and these tumors were examined for PD1 and Tim-3 expression.

For circulating T cell experiments, CD45+CD3+ T cell fractions were selected for marker determination using directly labeled anti-Tim-3 or PD-1 antibodies (commercially available from Biolegend). Tim-3+PD-1+ cells constituted the predominant fraction of circulating T cells in the blood of untreated animals. At the same time, T-cells in the blood of animals treated with CD20xCD3 bispecific antibody (Ab 1) showed smaller fractions of Tim-3+PD-1+ cells.

Tumor cells from untreated hosts were separated and stained for viability. FACS analysis was performed on viable single cells to sort out CD45+CD3+ cell fractions, which were then examined for Tim-3 or PD-1 expression.

We found that inhibitory Tim-3 and PD-1 receptors were expressed on CD3+ TIL in NSG B-cell lymphomas of untreated mice during the experiments described in Example 12B, and Tim-3+PD-1+ cells were the predominant fraction of tumor-infiltrating T- cells.

Example 18 CD3 x CD20 Bispecific Antibody Treatment Is More Efficient Compared to Anti-CD20+ Antibody in Raji Tumor-Developed NSG Mice

The efficacy of selected anti-CD3xCD20 bispecific antibodies in reducing established tumors in NSG mice was evaluated. NSG mice (NOD/LtSz-scid/IL2RYnull mice; Jackson Laboratories) were subcutaneously implanted with Raji tumor cells (2x106) and human PBMCs (5x105) (on day -14) (similar to example 12B).

CD20xCD3 bispecific Ab1 (at 0.4 mg/kg; 2x/week, ip) was comparable to CD19xCD3 BiTE (at 0.5 mg/kg; 5x/week, i.v.) (Fig. 23 ) and was superior to rituximab therapy (8 mg/kg; 5x/week, ip) (Fig. 24) in suppressing advanced Raji tumors.

- 55 039429

Example 19 Treatment of CD3 x CD20 melanoma with a bispecific antibody

Researchers have found that some subpopulations of melanoma cancer in patients, such as CD20+ melanoma tumor cells, may exhibit tumor-initiating characteristics and an increased risk of disease recurrence (Pinc et al. Mol Ther. 20(5):1056-1062, 2012, epub Feb 21, 2012). The CD20xCD3 bispecific antibody Ab1 showed high activity against other CD20-expressing tumor cells, as it significantly retarded the growth of hCD20-transduced B16F10.9 (B16F10.9/CD20) tumors in immunocompetent mice.

Example 20 Phase 1 clinical study to investigate the safety and tolerability of an anti-CD20xCD3 bispecific monoclonal antibody in patients with CD20+ B cell malignancies previously treated with a CD20-targeted antibody

Research objectives and study overview

The primary goal of this study is to evaluate the safety, tolerability, and dose-limiting toxicity (DLT) of an intravenously administered anti-CD20xCD3 bispecific monoclonal antibody. The anti-CD20xCD3 bispecific antibody used in the present study was the antibody referred to herein as Ab1.

The secondary objectives of the study are: (a) to characterize the pharmacokinetic (PK) profile of Ab1; (b) assessing the immunogenicity of Ab1; (c) study of preliminary antitumor activity of Ab1 administered to patients with CD20+ B cell malignancies (non-Hodgkin's lymphoma [NHL] and chronic lymphocytic leukemia [CLL]) who have previously received anti-CD20 antibody therapy.

The specific exploratory goals of the study were to evaluate biomarkers that might correlate with the mechanism of action, observed cytotoxicity, and potential antitumor activity, including, but not limited to: cytokine analysis, peripheral blood B and T cell subset phenotyping and immune phenotyping, as well as changes in gene expression in peripheral blood.

Study Design

An open, multicentre, dose escalation study was conducted with the anti-CD20xCD3 monoclonal antibody Ab1 administered via BB infusion. Patients were assigned a specific dosage level (LD) consisting of an initial dose followed by higher doses for the second and subsequent administrations. Patients were selected based on indication (NHL or CLL). Each LE corresponds to 2 groups (one for each indication) with 3-6 patients per group.

Patients who initially show clinical improvement but then relapse or progression of the disease may be re-treated with Ab1 at the highest SL considered tolerable during relapse or progression.

Patients already selected for the study underwent screening procedures to determine their eligibility for 28 days prior to initial administration of Ab1. Patients were selected sequentially based on indication (NHL or CLL) to confirm compliance with the sponsor until each group was filled according to the protocol.

Separate independent groups were created for NHL and CLL, with dose escalation at each DL. Each UD consisted of an initial dose and a second and subsequent dose that exceeded the initial dose, provided that the initial dose was tolerable.

The dose escalation followed the traditional 3+3 dose escalation design. Based on the observed toxicity, three to 6 patients were planned to be included in the group.

After completion of the dose escalation phase and after determining the recommended dose for additional study, it was planned to create 3 expansion groups with insensitive NHL, aggressive NHL and CLL, 10 patients in each expansion group.

For the first UD, a 48-hour waiting period is required between doses in the initial study for the first 3 patients within the same indication. The following patients at the first UD are not treated on the same day, regardless of the indication. In the following groups, provided there is no unexpected toxicity in the previous groups or within the group, the initial infusions for the first 3 patients should be given at least 24 hours apart.

Once selection, treatment, and follow-up period of EBRT is completed for each patient population, the opening of the next LD groups for selection (or expansion of the current DL groups) is determined after review of the safety data by the sponsor and investigator(s). The follow-up period for ESWL is defined as the first 28 days of treatment, which in this study corresponds to the induction period. During induction, patients are treated with 4 weekly administrations of Ab1.

An individual patient must receive at least 2 first injections of Ab1 (week 1, day 1 and week 2, day 1) in order to evaluate SWL. In addition, the patient must be observed for at least 28 days after the first injection and at least 21 days after the second injection.

Study duration

The treatment period is 6 months. Patients are initially treated with up to 9 doses of Ab1-4 weekly doses over a 4 week induction period followed by 5 doses administered monthly over a 5 month maintenance period. The observation period is 6 months.

Patient population

Up to 84 patients may be required in dose escalation arms for both indications (NHL and CLL) during the escalation phase (assuming 6 patients in each arm from EL1 to EL7), an additional 36 patients may be included by adding groups EL8-LE10 and up to 30 additional patients (20 NHL [10 each for refractory and aggressive NHL] and 10 CLL) may be required in expanded groups, for a total of 150 patients.

Patients must be those who have confirmed CD20+ B cell malignancies, the disease must be refractory to previous therapy, who have previously received CD20-targeted antibody therapy, for whom there are no standard treatment options, and for whom anti-CD20 antibody treatment may be appropriate .

Inclusion criteria for the study are as follows: (1) the presence of a confirmed malignant CD20+ B-cell formation, the disease must be refractory to previous therapy, the absence of standard treatment options, and the advisability of treatment with an anti-CD20 antibody; (2) prior anti-CD20 antibody therapy; (3) all patients (B-cell NHL and CLL) must have at least a bidimensionally measurable lesion >1.5 cm) confirmed by CT scan; (4) CLL patients must have white blood cells (WBCs)) <200x109; (5) age >18 years; (6) Eastern United Cancer Group (ECOG) General Condition <1; (7) life expectancy of at least 6 months; (8) normal bone marrow function confirmed by: (a) platelet count >75 x 109/l, (b) hemoglobin level >9 g/dl, and (c) ANC >1 x 109/l; (9) normal organ function; (10) agreeing to undergo mandatory prior tumor biopsy if, from the perspective of the investigator, the patient has a measurable lesion that can be biopsied without significant risk to the patient; (11) willingness and ability to comply with clinic visits and study-related procedures; and (12) providing signed informed consent.

The criteria for exclusion from the study are as follows:

(1) primary central nervous system (CNS) lymphoma or known or suspected CNS involvement of non-primary CNS NHL;

(2) history or current relevant CNS pathology such as (a) epilepsy, paroxysm, paresis, aphasia, apoplexy, severe brain lesions, cerebellar disease, organic brain syndrome, psychosis, or (b) evidence of inflammatory lesions and/or vasculitis with MRI of the head;

(3) standard anti-lymphoma chemotherapy (non-biologic) or radiation therapy within 28 days prior to first administration of study drug;

(4) previous therapy with blinatumomab;

(5) transplantation of allogeneic stem cells;

(6) treatment with rituximab, alemtuzumab, or other research or commercial biological agent within 12 weeks prior to first administration of study drug [CAUTION: for patients with aggressive lymphoma who require immediate treatment, the washout period may be reduced to 28 days];

(7) immunosuppressive therapy (other than biologic) within 28 days of first administration of study drug;

(8) treatment with an experimental non-biological agent within 28 days of the first administration of study drug;

(9) history of allergic reactions caused by compounds with similar chemical or biological composition to the study drug;

(10) history of hypersensitivity to any compound from the group of tetracycline antibiotics;

(11) history of malignancies other than B-cell malignancies within 5 years prior to study entry, excluding resected/removed basal or squamous cell carcinoma of the skin or carcinoma in situ of the cervix;

(12) the presence of a bacterial, viral, fungal or mycobacterial or other infection or any serious episode of infection requiring hospitalization or IV anti-infective treatment within 4 weeks prior to first administration;

(13) evidence of a serious concomitant disease or condition that may interfere with the conduct of the study or place the patient at significant risk;

(14) current systemic corticosteroid treatment, excluding corticosteroid use for other (non-neoplastic and non-immunosuppressive) indications, up to a maximum of 5 mg/day of prednisone or its equivalent;

- 57 039429 (15) human immunodeficiency virus (HIV) infection or chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection;

(16) presence of hypersensitivity to allopurinol and rasburicase;

(17) pregnant or breastfeeding women;

(18) sexually active men or women of childbearing potential who do not wish to use adequate contraception during the study.

Treatment Options

Ab1 was supplied as a liquid in sterile disposable vials. Each vial contains a withdrawal volume of 1 ml of Ab1 at a concentration of 2 mg/ml. Detailed preparation and administration instructions will be provided locally in pharmaceutical manuals.

The initial dose of Ab1 at dosage level (LD) 1 was 30 μg; doses may be increased up to 8000 micrograms when reaching an EL10. Ab1 doses were administered to enrolled patients on an outpatient basis by BB infusion over at least 60 minutes. Dosing levels are given in table. 23.

Table 23. Dose escalation and groups for patients with NHL and CLL

Dosing level Initial dose (mcg) 2nd and subsequent dose (mcg) Indication: NHL NHL: (P) Indication: HLL HLL: (P) UD-1 ten thirty Group 1H* 3-6 Group IX* 3-6 LE1 thirty 100 Group 1H 3-6 Group IX 3-6 LE 2 100 300 Group 2H 3-6 Group 2X 3-6 UDZ 300 1000 ZN group 3-6 ZX Group 3-6 LE 4 1000 2000 Group 4H 3-6 Group 4X 3-6 LE 5 2000 3000 Group 5H 3-6 Group 5X 3-6 LE 6 3000 4000 Group 6H 3-6 Group 6X 3-6 LE 7 4000 5000 Group 7H 3-6 Group 7X 3-6 LOD8** 5000 6000 Group 8H 3-6 Group 8X 3-6 LE9** 6000 7000 Group 9H 3-6 Group 9X 3-6 UDU** 7000 8000 UN Group 3-6 YUH group 3-6

*N=NHL;

X=HLL;

EL=Dosage Level ** Dosage levels 8-10 can be opened at completion of EL7, MTD is not determined, and PK, PD, and clinical parameters suggest that the optimal biological dose may be higher than originally thought.

Patients received Ab1 weekly for a 4-week induction period, followed by monthly doses of 5 additional doses, at the dosage appropriate for the group.

Endpoints

The primary endpoint is safety (particularly adverse events [AEs] and SWL) to determine the maximum tolerated dose (MTD) and/or optimal biological dose (OBD) as the recommended dose for phase 2 (RDF2) Ab1.

Secondary endpoints are:

(a) pharmacokinetics: Ab1 concentration; (b) immunogenicity: anti-Ab1 antibodies; (c) antitumor activity, including overall response rate (ORR), progression-free survival (PFS) and overall survival (OS), as well as minimal residual disease (MRD) for patients with CLL. For TMR, tumor response is assessed according to the Revised Response Criteria for Malignant Lymphomas of the National Cancer Institute International Working Group (NCI-WG) and according to the CLL Diagnosis and Treatment Guidelines of the International Workshop on Chronic Lymphocytic Leukemia.

The exploratory endpoints of the present study included pharmacodynamic (PD) measurements including: (a) B cell and T cell subgroups and phenotype, (b) circulating cytokine levels, and (c) C-reactive protein (CRP).

Procedures and assessments

Control procedures include MRI of the brain, electrocardiogram (ECG), testing for human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV), and blood clotting.

Safety procedures include history, physical examination, assessment of B-symptoms, evaluation of general condition, clinical laboratory tests, vital signs, AEs, and concomitant medications.

Efficacy procedures include tumor evaluation, including CT scan, 18F-fluorodeoxyglucose positron emission tomography (PET-FDG), bone marrow aspirates and biopsy, lymph node and/or tumor biopsy, and peripheral blood samples (CLL patients only).

Blood samples for PK assessment and anti-drug antibodies (APP) were obtained from enrolled patients.

Biomarker samples were obtained from enrolled patients to monitor changes in cytokine production, serum levels of pro-inflammatory cytokines, and changes in lymphocyte subgroups and activation status. In addition, these samples allow for tumor analysis or somatic genetic analysis for variations that affect the clinical course of the underlying disease or modulate the side effects of treatment.

The present invention is not intended to be limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art upon examination of the foregoing description and accompanying figures. Such modifications are intended to be within the scope of the appended claims.

Claims (12)

1. A method of treating or ameliorating a CD20-positive B cell cancer in a subject, comprising administering a dose escalation protocol that reduces the effect of a cytokine cascade, wherein the dose escalation protocol comprises administering a first dose of a bispecific antibody and then administering a second dose of said a bispecific antibody, wherein said second dose is greater than said first dose, wherein the bispecific antibody comprises a first antigen-binding domain that binds human CD3, a second antigen-binding domain that binds human CD20, and a heavy chain constant region associated with each of the first and second antigen-binding domains where the heavy chain constant region contains: (a) the amino acid sequence of the lower hinge region of human IgG2 containing PCPAPPVA (SEQ ID NO: 52) from positions 228 to 236 (EU numbering); (b) the amino acid sequence of the upper hinge region of human IgG1 or human IgG4 from positions 216 to 227 (EU numbering); (c) the amino acid sequence of the CH2 domain of human IgG4 from positions 237 to 340 (EU numbering); (d) CH1 domain of human IgG1 and CH3 domain of human IgG1 or CH1 domain of human IgG4 and CH3 domain of human IgG4; wherein the bispecific antibody exhibits higher binding affinity for human FcyRIIA compared to human FcyRIIB, and exhibits little or no detectable binding affinity for human FcyRI and human FcyRIII as measured by surface plasmon resonance. 2. The method of claim 1, wherein the B cells are pre-B lymphocytes, mature B lymphocytes, or B-cell non-Hodgkin's lymphoma cells. 3. The method of claim 1, wherein the subject is selected based on the presence of residual cancer. 4. Method according to any one of claims 1 to 3, characterized in that the cancer is lymphoma or leukemia. 5. The method according to any one of claims 1 to 4, characterized in that the cancer is selected from the group consisting of: follicular lymphoma, B-cell chronic lymphocytic leukemia, B-cell lymphoblastic lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma , marginal zone lymphoma, mantle cell lymphoma, hairy cell leukemia, and Burkitt's lymphoma. 6. Method according to any one of claims 1 to 5, characterized in that the first dose is up to 1000 μg. 7. The method of any one of claims 1 to 6, wherein the subject has a tumor that is resistant or not fully responsive to (a) anti-CD20 monospecific antibody therapy alone or (b) rituximab monotherapy. 8. The method according to any one of claims 1 to 7, wherein the subject has been treated with an anti-CD20 monospecific antibody for at least 1 day to 1 year prior to administration of the bispecific antibody. 9. A method according to any one of claims 1 to 8, wherein the heavy chain constant region comprises: (a) amino acid sequence of the EPKSCDKTHTCPPCPAPPVA chimeric hinge region - 59 039429 (SEQ ID NO: 53); or (b) the amino acid sequence of the chimeric hinge region ESKYGPPCPPCPAPPVA (SEQ ID NO: 54). 10. The method according to any one of claims 1 to 9, characterized in that: (a) the first antigen-binding domain contains the amino acid sequence of the heavy chain variable region (HCVR) containing SEQ ID NO: 10; (b) the first antigen-binding domain comprises a light chain variable region (LCVR) amino acid sequence comprising SEQ ID NO: 18; (c) the second antigen-binding domain contains the amino acid sequence of the heavy chain variable region (HCVR) containing SEQ ID NO: 2; (d) the first antigen-binding domain that specifically binds human CD3 comprises a heavy chain variable region (HCVR) amino acid sequence comprising SEQ ID NO: 10 and a light chain variable region amino acid sequence (LCVR) comprising SEQ ID NO: 18; (e) a second antigen-binding domain that specifically binds human CD20 comprises a heavy chain variable region (HCVR) amino acid sequence comprising SEQ ID NO: 2 and a light chain variable region amino acid sequence (LCVR) comprising SEQ ID NO: 18; (f) the first antigen binding domain (A1) contains three heavy chain complementarity determining regions (HCDR1, HCDR2, HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, LCDR3), wherein (i) A1-HCDR1 is the amino acid sequence of SEQ ID NO: 12; (ii) A1-HCDR2 is the amino acid sequence of SEQ ID NO: 14; (iii) A1-HCDR3 is the amino acid sequence of SEQ ID NO: 16; (iv) A1-LCDR1 is the amino acid sequence of SEQ ID NO: 20; (v) A1-LCDR2 is the amino acid sequence of SEQ ID NO: 22; and (vi) A1-LCDR3 is the amino acid sequence of SEQ ID NO: 24; or (g) a second antigen binding domain (A2) that specifically binds human CD20 contains three heavy chain complementarity determining regions (HCDR1, HCDR2, HCDR3) and three light chain complementarity determining regions (LCDR1, LCDR2, LCDR3), wherein (i) A2-HCDR1 is the amino acid sequence of SEQ ID NO: 4; (ii) A2-HCDR2 is the amino acid sequence of SEQ ID NO: 6; (iii) A2-HCDR3 is the amino acid sequence of SEQ ID NO: 8; (iv) A2-LCDR1 is the amino acid sequence of SEQ ID NO: 20; (v) A2-LCDR2 is the amino acid sequence of SEQ ID NO: 22; and (vi) A2-LCDR3 is the amino acid sequence of SEQ ID NO: 24. 11. The method according to any one of claims 1 to 9, wherein the first antigen binding domain (A1) that specifically binds human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2, A1HCDR3) and three light complementarity determining regions. chains (A1-LCDR1, A1-LCDR2, A1LCDR3), and the second antigen-binding domain (A2), which specifically binds human CD20, contains three heavy chain complementarity-determining regions (A2-HCDR1, A2-HCDR2, and A2-HCDR3) and three complementarity of the light chain region (A2-LCDR1, A2-LCDR2 and A2LCDR3); wherein (i) A1-HCDR1 is the amino acid sequence of SEQ ID NO: 12; (ii) A1-HCDR2 is the amino acid sequence of SEQ ID NO: 14; (iii) A1-HCDR3 is the amino acid sequence of SEQ ID NO: 16; (iv) A1-LCDR1 is the amino acid sequence of SEQ ID NO: 20; (v) A1-LCDR2 is the amino acid sequence of SEQ ID NO: 22; (vi) A1-LCDR3 is the amino acid sequence of SEQ ID NO: 24; (vii) A2-HCDR1 is the amino acid sequence of SEQ ID NO: 4; (viii) A2-HCDR2 is the amino acid sequence of SEQ ID NO: 6; (ix) A2-HCDR3 is the amino acid sequence of SEQ ID NO: 8; (x) A2-LCDR1 is the amino acid sequence of SEQ ID NO: 20; (xi) A2-LCDR2 is the amino acid sequence of SEQ ID NO: 22; and (xii) A2-LCDR3 is the amino acid sequence of SEQ ID NO: 24. 12. The method of claim 11, wherein the first antigen-binding domain that specifically binds to human CD3 comprises (i) a heavy chain variable region (HCVR) which is the amino acid sequence of SEQ ID NO: 10, and (ii) a light chain variable region chain (LCVR), which is the amino acid sequence of SEQ ID NO: 18; and a second antigen-binding domain that specifically binds human CD20 contains (iii) a heavy chain variable region (HCVR) which is an amino acid sequence - - 60 039429 ity of SEQ ID NO: 2, and a light chain variable region (LCVR), which is the amino acid sequence of SEQ ID NO: 18. Construction of the chimeric hinge region Upper hinge region Lower hinge region lgG1, EU numbering 216 217 213 219 220 221 222 223 224 225 226 227 igGi, Kabat numbering 226 227 228 232* 229* 233 ^L 230^ 294* 232* 233 236 IgGl E R To S Me and 0 To T H T FROM sill igG2 R FROM R BUT R R V BUT l ₆ G4 Е S К Y G (ill Р р with II lgG4, Numbering , EU 216 217 218 219* 220 ^ь 224 225 226 227 230 237 238 239 240 Numbering Kabat 2M 242 243 244 245 24V 247 248 - means no corresponding number - indicates the absence of the corresponding amino acid ^and the numbering is in accordance with the latest update of the IMGT Scientific Chart, (IMGT*, International Immunogenetic Information System®, httpV/illmimgtorg/IMGTSciectificChart/NLimber^^ Created - May 17, 7001, last updated: January 10, 2013. ^b Numbering according to the EU index, copied from Kabat (Kabat, FA el al, Sequences of Proteins of Immunological interest 5th ed US Department of Health and Human Services, NIH publication No. ^91-3242 (1991) Fig. one 10 20 30 40 5060 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS *—CHI Superior/inferior arar region—* CH2—* 70 80 90 100 110120 GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 130 140 150 160 170180 PSVFLFPPKP KDTLMISRTP EVTCVWDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN CH3- ^* 190 200 210 220 230240 STYRWSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 250 260 270 280 290300 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 310 320330 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK Heavy chain constant region of human IGHG1 UniProtKB/Swiss-Prot Accn. No. P01857 (SEQ ID NO:45) Fig. 2 10 20 30 40 5060 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS *—CHI Upper/lower hinge region—»CH2- 70 80 90 100 110120 GLYSLSSWT VPSSNFGTQT YTCNVDHKPS NTKVDKTVHH KCCVECPPCP APPVAGPSVF 130 140 150 160 170180 LFPPKPKDTL MISRTPEVTC VWDVSHEDP EVQFNWYVDG VEVHNAKTKP REEQFNSTFR CH3—> 190 200 210 220 230240 WSVLTWHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL PPSREEMTKN 250 260 270 280 290300 QVSLTCLVKG FYPSDISVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT VDKSRWQQGN 310320 VFSCSVMHEA LHNHYTQKSL SLSPGK Heavy chain constant region of human IGHG2 UniProtKB/Swiss-Prot Accn. No. P01859 (SEQ ID NO:46) Fig. 3 - 61 039429 10 20 30 40 5060 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS *— CHI Upper/lower pivot region—>CH2—> 70 80 90 100 110120 GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVHS KYGPPCPSCP APEFLGGPSV 130 140 150 160 170180 FLFPPKPKDT LMISRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY CH3—> 190 200 210 220 230240 RWSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK 250 260 270 280 290300 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 310320 NVFSCSVMHE ALHNHYTQKS LSLSLGK Heavy chain constant region of human IGHG4 UniProtKB/Swiss-Prot Accn. No. P01861 (SEQ ID NO:47) Fig. four Fig. 5A,B - 62 039429 Fig. 6A,B Treatment with Abi or Control in NSG mice implanted with Raji tumor cells and PBMCs Control: Solvent Ratio PBMC: tumor 1:4 Control: Control AL5, 0.4 mg/kg PBMC:tumor 1:4 3000η CD20XCD3: AL1.0.4 mg/kg PBMC: tumor 1:4 2000(5/5) 10005 10 15 20 25 Days after implantation - 63 039429 Treatment of NSG mice implanted with Raji tumor cells without PBMC control with AI 5000 _Ί 40003000Control: Without MCPC AI, 0.4 mg/kg MCPC 0:4 about 5 10 15 20 Days after implantation D Treatment of NSG mice implanted with Raji and PBMC tumor cells with AI 30001 CD20XCD3: AM, 0.04 mg/kg PBMCgotumor 1:4 ZOI η CD20XCD3: AL1.0.004 mg/kg PBMC: tumor 1:4 200020001000(W 5 10 15 20 Days after implantation * about £8 1000 (5/5) 5 10 1 5 20 25 Days after implantation Fig. 7A-F AT Fig. 8A,B - 64 039429 Treatment of developed -200-400 mm ³ tumors in NSG mice CD3xCD20 bispecific antibody Days after treatment Fig. 9 Treatment of mature -500-900 mm ³ tumors in NSG CD3xCD20 mice with a bispecific antibody 0 10 20 30 days after processing Fig. ten Fig. eleven - 65 039429 CD3xCD20 bispecific antibodies mediate long-term decline - Placebo Antibody 4 (1.0 mg/kg) Antibody 1 (1.0 mg/kg) Fig. 12 CD3xCD20 bispecific antibodies show a decrease in peripheral blood B cells at low dosage levels Days after dosing Antibody 4 (0.01 mg/kg) Antibody 1 (0.01 mg/kg) Antibody 4 (0.001 mg/kg) - Antibody 1 (0.001 mg/kg) Fig. 13 Recovery of B cells correlates with disappearance of detectable levels of circulating bispecific antibodies 1 mg/kg (Antibody 1) Time (day) Fig. fourteen - 66 039429 Recovery of B cells correlates with disappearance of detectable levels of circulating bispecific antibodies 1 mg/kg (Antibody 2) 100 -*-106876 7.50 -14 -7 0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 Time (day) Fig. fifteen Fig. 16 CD3xCD20 bispecific antibodies show ability to down-regulate B cells in lymphoid organs Spleen Fig. 17 - 67 039429 CD3xCD20 bispecific antibodies induce T cell proliferation in vitro Fig. eighteen CD3xCD20 bispecific antibodies mediate cell killing by activated T cells in a tn vitro bioassay Fig. 19A,B Fig. 20A,B - 68 039429 T cell activation markers are induced by treatment with bispecific AL Upregulation of CD69 in a 48-hour „„„ cytotoxicity assay targeting Bl6F10.9 CD20 cells ΊΟΟ-ι Antibody [M] Fig. 21 CD3xCD20 bispecific antibodies induce clustering of T cells with lgGI-Isohyp target cells 0 hg ON-A 'T target cells A B Fig. 22A,B Treatment of NSG mice with advanced Raji tumors NS Days after । treatment - ·- Control - Base solution Control AB 6 - 0.4 mg/kg; 2x/week - F-Ab 1 - 0.4 mg/kg; 2x/week - F-Rituximab - 8 mg/kg; 5x/week - F-CD19xCD3 BiTE - 0.5 mg/kg; 5x/week **** - in comparison with the control base solution; No. - compared with the control AL6; @ - but compared with ritukeimab; ns - ns significant The data represent pooled data for n-4-6 mice on ipynny. Data are expressed as mean (SPS) and were analyzed using analysis of variance (ANOVA) and post hoc tests to examine significant effects (Tioki's two-way ANOVA). Two mice from the CD 19 BiTE group and one mouse from the AL group were excluded from this pooled graph due to early death in order to analyze the data using a two-way ANOVA. Fig. 23 - 69 039429 Treatment of NSG mice with advanced Raji tumors Days after treatment Control - Base solution -I- Control AB 6 - 0.4 mg/kg; 2x/week TK- AB 1 - 0.4 mg/kg; 2x/week Rituximab - 8 mg/kg; 5x/week **** - compared with the control stock solution; /W/ - compared with the control AL6; @ - compared to rituximab The data represent pooled data for n=4-6 mice per group. Data are expressed as mean (SPS) and were analyzed using analysis of variance (ANOVA) and post hoc tests to examine significant effects (Tukey's two-way ANOVA). One large AN mouse was excluded from this pooled chart due to early death in order to analyze the data using two-way AMOUA. Fig. 24 Treatment of hCD3 mice with implanted CD20/B16F.9 tumors Days after starting therapy Control - Base Solution Control AB 6 - 0.4 mg/kg - Ab1 - 0.4 mlLg - AB 1 - 0.004 mg/kg **** - and compared with the baseline expand - w compared with cochrolone Abb mice on ipywiy. Data are expressed as mean (MAS) and were analyzed using an analysis of variance (ANOVA) and a superior test for learning significant affects (Gyoka's two-factor ANOVA). - Control - Stock solution Control AB 6 - 4 mg/kg -g» AI - 4 mg/kg mv» AB 1 - 0.4 mg/kg * -110 compared with the baseline extended and co-corrected antibody Data ipyuny are summarized data of d.sh n-5 mice on ipyuny. Dan™ gs are expressed as mean (ATS) and were analyzed using analysis of variance (ANOVA).