Bispecific antibody or antigen-binding fragment thereof, its use, as well as pharmaceutical composition.

Abstract

The present invention relates to anti-mucin 16 antibodies: bispecific antibodies (bsabs) that bind to both muc16 and CD3 and activate T cells via the CD3 complex in the presence of tumors expressing muc16; human IgG antibodies that bind to both CD3 and human muc16 (monospecific antibodies); drug-anti-muc16 antibody conjugates that inhibit tumor growth in vivo; antibodies are useful for the treatment of various cancers, including ovarian cancer.

Description

Bispecific antibody or antigen-binding fragment thereof, its use, as well as pharmaceutical composition. REFERENCE TO A LISTING OF SEQUENCES

[001] This request incorporates the Listing as a reference. Sequences submitted in Machine Readable Form as file 10295WO01-Sequence.txt, created on September 22, 2017 and containing 893,983 bytes. FIELD OF THE INVENTION

[002] The present invention relates to antibodies and antigen-binding fragments thereof, which are specific for mucin 16 (MUC16), and to methods of using them. The present invention also relates to bispecific antigen-binding molecules that bind to MUC16 and CD3, and to methods of using them. The present invention further relates to antibody-drug conjugates comprising an anti-MUC16 antibody or a fragment thereof and a therapeutic agent (for example, a cytotoxic agent). BACKGROUND

[003] Mucin 16 (MUC16), also known as cancer antigen 125, carcinoma antigen 125, carbohydrate antigen 125, or CA-125, is a highly glycosylated integral membrane glycoprotein with a single transmembrane domain that is highly expressed in ovarian cancer. MUC16 consists of three main domains: an N-terminal extracellular domain, a large tandem repeat domain interspersed with sea urchin spermatozoa, enterokinase, and agrin (SEA), and a carboxyl-terminal domain comprising a segment of the transmembrane region and a short cytoplasmic tail. Proteolytic cleavage results in the spillage of much of the extracellular portion of MUC16 into the bloodstream. MUC16 is Petition 870260046565, dated 05 / 15 / 2026, page 10 / 435 MUC16 2 / 202 is overexpressed in cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, cervical adenocarcinoma, and gastric tract adenocarcinoma, including intrahepatic cholangiocarcinoma mass formation, and in diseases and conditions including inflammatory bowel disease, liver cirrhosis, heart failure, peritoneal infection, and abdominal surgery (Haridas, D. et al., 2014, FASEB J., 28:4.183-4.199). Expression in cancer cells has been shown to protect tumor cells from the immune system (Felder, M. et al., 2014, Molecular Cancer, 13:129). Modes for treating ovarian cancer using antibodies to MUC16 have been investigated. Oregovomab and abgovomab are anti-MUC16 antibodies that have had limited success. (Felder, supra, Das, S. and Batra, SK 2015, Cancer Res. 75:4.660 to 4.674.)

[004] CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed from the dimeric association of two of four different chains: epsilon, zeta, delta, and gamma. The dimeric arrangements of CD3 include gamma / epsilon, delta / epsilon, and zeta / zeta. Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the binding of peptide-laden MHC molecules to the TCR. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. Additionally, bispecific antibodies capable of binding to both CD3 and a target antigen have been proposed for therapeutic uses involving T cell immune responses targeting tissues and cells expressing the target antigen.

[005] Molecules that target MUC16, including antibody-drug conjugates, as well as molecules that bind to Petition 870260046565, dated 05 / 15 / 2026, p. 11 / 435 3 / 202 Bispecific antigens that bind to both MUC16 and CD3 would be useful in therapeutic settings where specific targeting and T cell-mediated killing of MUC16-expressing cells are desired. BRIEF SUMMARY OF THE INVENTION

[006] In a first aspect, the present invention provides antibodies and their antigen-binding fragments that bind to human MUC16. The antibodies according to this aspect of the invention are useful, among other things, for targeting cells expressing MUC16. The present invention also provides bispecific antibodies and their antigen-binding fragments that bind to human MUC16 and human CD3. Bispecific antibodies according to this aspect of the invention are useful, among other things, for targeting CD3-expressing T cells and for stimulating T cell activation, for example, under circumstances where T cell-mediated death of MUC16-expressing cells is beneficial or desirable. For example, bispecific antibodies can target CD3-mediated T cell activation to specific MUC16-expressing cells, such as ovarian tumor cells.

[007] The exemplary anti-MUC16 antibodies of the present invention are listed in Tables 1 and 2 herein. Table 1 presents the amino acid sequence identifiers of the heavy chain variable regions (HCVRs), light chain variable regions (LCVRs), heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-MUC16 antibodies. Table 2 presents the nucleic acid molecule sequence identifiers of HCVRs, LCVRs, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the exemplary anti-MUC16 antibodies. Petition 870260046565, dated 05 / 15 / 2026, page 12 / 435 4 / 202

[008] The present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence among the same having at least 90%, at least 95%, at least 98% or at least 99% sequence identity therewith.

[009] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0010] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCVR / LCVR amino acid sequence pair (HCVR / LCVR) comprising any one of the HCVR amino acid sequences listed in Table 1 paired with any one of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCVR / LCVR amino acid sequence pair contained in any one of the exemplary anti-MUC16 antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is of SEQ ID NOs: 18 / 26 (e.g., H1H8767P).

[0011] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a CDR1 heavy chain (HCDR1) comprising a Petition 870260046565, dated 05 / 15 / 2026, p. 13 / 435 5 / 202 amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0012] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a CDR2 heavy chain (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0013] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a CDR3 heavy chain (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0014] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0015] The present invention also provides antibodies, or Petition 870260046565, dated 05 / 15 / 2026, p. 14 / 435 6 / 202 antigen-binding fragments thereof, comprising a CDR2 light chain (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0016] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0017] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present invention provides antibodies, or antigen-binding fragments thereof, comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-MUC16 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is SEQ ID NOs: 24 / 32 (e.g., H1H8767P).

[0018] The present invention also provides antibodies, or antigen-binding fragments thereof, comprising a Petition 870260046565, dated 05 / 15 / 2026, p. 15 / 435 7 / 202 set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3) contained in any of the exemplary anti-MUC16 antibodies listed in Table 1. In certain embodiments, the amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2LCDR3 are selected from the group consisting of SEQ ID NOs: 2022-24-28-30-32 (e.g., H1H8767P).

[0019] In a related embodiment, the present invention provides antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence pair as defined by any of the exemplary anti-MUC16 antibodies listed in Table 1. For example, the present invention includes antibodies, or antigen-binding fragments thereof, comprising the amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 contained in an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ. ID NOs: 18 / 26 (e.g., H1H8767P). The methods and techniques for identifying CDRs in the amino acid sequences of HCVR and LCVR are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein.Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Broadly speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a consensus between the Kabat and Chothia approaches. See, for example, Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927. Petition 870260046565, dated 05 / 15 / 2026, p. 16 / 435 8 / 202 to 948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:92.68 to 9.272 (1989). Public databases are also available to identify CDR sequences within an antibody.

[0020] The present invention also provides nucleic acid molecules encoding anti-MUC16 antibodies or portions thereof. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same.

[0021] The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same.

[0022] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence among those that have Petition 870260046565, dated 05 / 15 / 2026, p. 17 / 435 9 / 202 at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0023] The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same.

[0024] The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same.

[0025] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same.

[0026] The present invention also provides acid molecules Petition 870260046565, dated 05 / 15 / 2026, p. 18 / 435 10 / 202 nucleic acids encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same.

[0027] The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same.

[0028] The present invention also provides nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the amino acid sequence set HCDR1-HCDR2-HCDR3 is defined by any of the exemplary anti-MUC16 antibodies listed in Table 1.

[0029] The present invention also provides nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), wherein the set of amino acid sequences LCDR1-LCDR2-LCDR3 is as defined by any of the exemplary anti-MUC16 antibodies listed in Table 1.

[0030] The present invention also provides acid molecules Petition 870260046565, dated 05 / 15 / 2026, p. 19 / 435 11 / 202 nucleic acids encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence from any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence from any of the LCVR amino acid sequences listed in Table 1.In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence among the same that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the same. In certain embodiments according to this aspect of the invention, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-MUC16 antibody listed in Table 1.

[0031] The present invention further provides recombinant expression vectors capable of expressing a polypeptide comprising a variable region of the heavy or light chain of an anti-MUC16 antibody. For example, the present invention includes recombinant expression vectors comprising any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR and / or CDR sequences, as shown in Table 1. Furthermore, the scope of the present invention encompasses host cells into which such vectors have been introduced, as well as Petition 870260046565, dated 05 / 15 / 2026, page 20 / 435 12 / 202 methods for producing antibodies or portions thereof by cultivating host cells under conditions that permit the production of antibodies or antibody fragments and recovering the antibodies or antibody fragments thus produced.

[0032] The present invention includes anti-MUC16 antibodies that have a modified glycosylation pattern. In some embodiments, a modification to remove undesirable glycosylation sites or an antibody devoid of a fucose chemical moiety present in the oligosaccharide chain may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, a glycosylation modification may be produced in order to modify complement-dependent cytotoxicity (CDC).

[0033] In another aspect, the invention provides a pharmaceutical composition comprising a recombinant human antibody, or fragment thereof, that specifically binds to MUC16 and a pharmaceutically acceptable carrier. In another related aspect, the invention presents a composition consisting of a combination of an anti-MUC16 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-MUC16 antibody. Additional combination therapies and co-formulations involving the anti-MUC16 antibodies of the present invention are disclosed elsewhere in this document.

[0034] In another aspect, the invention provides therapeutic methods for targeting / destroying tumor cells expressing MUC16, using an anti-MUC16 antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-MUC16 antibody of the invention to an individual. Petition 870260046565, dated 05 / 15 / 2026, page 21 / 435 13 / 202 which requires the same. In some cases, anti-MUC16 antibodies (or their antigen-binding fragments) can be used to treat cancer (e.g., ovarian cancer), or can be modified to be more cytotoxic by methods including, but not limited to, Fc domains modified to increase ADCC (see, for example, Shield et al. (2002) JBC 277: 26733), radioimmunotherapy, antibody-drug conjugates, or other methods to increase the efficiency of tumor ablation.

[0035] The present invention also includes the use of an anti-MUC16 antibody of the invention in the manufacture of a medicament for the treatment of a disease or disorder (e.g., cancer) related to or caused by cells expressing MUC16. In one aspect, the invention relates to a compound comprising an anti-MUC16 antibody or antigen-binding fragment or a bispecific MUC16xCD3 antibody, as disclosed herein, for use in medicine. In one aspect, the invention relates to a compound comprising an antibody-drug conjugate (ADC) as disclosed herein, for use in medicine.

[0036] In yet another aspect, the invention provides monospecific anti-MUC16 antibodies for diagnostic applications, such as, for example, imaging reagents.

[0037] In yet another aspect, the invention provides therapeutic methods for stimulating T cell activation using an anti-CD3 antibody or an antigen-binding portion of an antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody.

[0038] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment that binds to human mucin 16 (MUC16) with an equilibrium constant of Petition 870260046565, dated 05 / 15 / 2026, p. 22 / 435 14 / 202 binding dissociation (Kd) less than about 53 nM, as measured in a surface plasmon resonance assay at 25 °C. In yet another aspect, the present invention provides an isolated antibody or an antigen-binding fragment thereof that binds to human MUC16 with a dissociative half-life (0½) greater than about 15 minutes, as measured in a surface plasmon resonance assay at 25 °C.

[0039] The invention further provides an antibody or antigen-binding fragment that competes for binding to human MUC16 with a reference antibody comprising a pair of HCVR / LCVR amino acid sequences as presented in Table 1. In another aspect, the invention provides an antibody or antigen-binding fragment that competes for binding to human MUC16 with a reference antibody comprising a pair of HCVR / LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 74; 82 / 90; 98 / 106; 114 / 122; 130 / 138; 146 / 154; 162 / 170; 178 / 186; 194 / 394; 202 / 210; 218 / 226, 234 / 242; 250 / 1936; 258 / 266; 274 / 1936; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386.

[0040] The invention further provides an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereto binds to the same epitope in human MUC16 as a reference antibody comprising an HCVR / LCVR amino acid sequence pair as presented in Table 1. In another aspect, the antibody or antigen-binding fragment binds to the same epitope in human MUC16 as a reference antibody comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 74; 82 / 90; 98 / 106; 114 / 122; 130 / 138; 146 / 154; 162 / 170; 178 / 186; 194 / 394; 202 / 210; Petition 870260046565, dated 05 / 15 / 2026, p. 23 / 435 15 / 202 218 / 226, 234 / 242; 250 / 1936; 258 / 266; 274 / 1936; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386.

[0041] The invention further provides an isolated antibody or antigen-binding fragment thereof that binds to human MUC16, wherein the antibody or antigen-binding fragment comprises: the complementarity-determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence as presented in Table 1; and the CDRs of a light chain variable region (LCVR) having an amino acid sequence as presented in Table 1. In another aspect, the isolated antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a pair of HCVR / LCVR amino acid sequences selected from the group consisting of: 2 / 10; 18 / 26; 34 / 42; 50 / 58; 66 / 74; 82 / 90; 98 / 106; 114 / 122; 130 / 138; 146 / 154; 162 / 170; 178 / 186; 194 / 394; 202 / 210; 218 / 226, 234 / 242; 250 / 1936; 258 / 266; 274 / 1936; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386. In yet another aspect, the isolated antibody or antigen-binding fragment comprises domains of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, selected from the group consisting of: SEQ ID NOs: 4-6-8-12-14-16; 20-22-24-28-30-32; 36-38-40-44-46-48; 52-54-56-60-62-64; 68-70-72-76-78-80; 84-86-88-92-94-96; 100-102-104-108-110-112; 116-118-120-124-126-128; 132-134-136-140-142-144; 148-150-152-156-158-160; 164-166-168-172-174-176; 180-182-184-188-190-192; 196-198-200-396-398-400; 204-206-208-212-214-216; 220-222-224-228-230-232; 236-238-240-244-246-248; 252-254-256-1938-1940-1942; 260-262-264-268-270-272; 276-278-280-1938-1940-1942; 284-286-288-292-294-296; 300-302-304-308-310-312; 316-318-320-324-326-328; 332-334-336-340-342-344; 348-350-352-356-358-360; 364-366368-372-374-376; and 380-382-384-388-390-392. Petition 870260046565, dated 05 / 15 / 2026, p. 24 / 435 16 / 202

[0042] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to human MUC16, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 202, 218, 234, 250, 258, 274, 282, 298, 314, 330, 346, 362, and 378; and (b) a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10; 26; 42; 58, 74; 90; 106; 122; 138; 154; 170; 186; 210; 226, 242; 266; 290; 306; 322; 338; 354; 370; 386; 1936 and 394.In a further aspect, the isolated antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment comprises a pair of HCVR / LCVR amino acid sequences selected from the group consisting of: SEQ ID NOs: 2 / 10; 18 / 26; 34 / 42; 50 / 58, 66 / 74; 82 / 90; 98 / 106; 114 / 122; 130 / 138; 146 / 154; 162 / 170; 178 / 186; 194 / 394; 202 / 210; 218 / 226, 234 / 242;. 250 / 1936; 258 / 266; 274 / 1936; 282 / 290; 298 / 306; 314 / 322; 330 / 338; 346 / 354; 362 / 370; and 378 / 386.

[0043] The invention further provides an isolated antibody or antigen-binding fragment thereof that binds to human MUC16 within an epitope ranging from residue 428 to residue 481 of SEQ ID NO: 1902. In some cases, the isolated antibody or antigen-binding fragment interacts with amino acid residues 428 to 434, 429 to 434, 453 to 467, 459 to 467, 460 to 467 and / or 474 to 481 of SEQ ID NO: 1902. In some embodiments, the antibody or antigen-binding fragment interacts with amino acid residues 428 to 434, 429 to 434, 453 to 467, 459 to 467, 460 to 467 and 474 to 481 of SEQ ID NO: 1902. The invention further provides an isolated antibody or antigen-binding fragment thereof that binds to Petition 870260046565, dated 05 / 15 / 2026, p. 25 / 435 17 / 202 The invention provides an isolated antibody or antigen-binding fragment that binds to human MUC16 within an epitope ranging from residue 126 to residue 138 of SEQ ID NO: 1902. In some cases, the isolated antibody or antigen-binding fragment interacts with amino acid residues 126 to 131, 127 to 131, and / or 132 to 138 of SEQ ID NO: 1902. In some embodiments, the antibody or antigen-binding fragment interacts with amino acid residues 126 to 131, 127 to 131, and 132 to 138 of SEQ ID NO: 1902. The invention further provides an isolated antibody or antigen-binding fragment thereto that binds to human MUC16 within an epitope ranging from residue 357 to residue 369 of SEQ ID NO: 1902. In some cases, the isolated antibody or antigen-binding fragment interacts with the residues of amino acid 357 to 369, 358 to 366, 358 to 369 and / or 361 to 369 of SEQ ID NO: 1902. In some embodiments, the antibody or antigen-binding fragment interacts with amino acid residues 357 to 369, 358 to 366, 358 to 369 and 361 to 369 of SEQ ID NO: 1902.The invention further provides an isolated antibody or antigen-binding fragment thereof that binds to human MUC16 within one or more of the five proximal membrane SEA domains of human MUC16 (SEQ ID NO: 1899). The five proximal membrane SEA domains correspond to residues 13,791 to 14,451 of SEQ ID NO: 1899. In some cases, the antibody or antigen-binding fragment binds to a KD lower than about 60 nM as measured in a surface plasmon resonance assay at 25 °C. In some embodiments, the antibody or antigen-binding fragment binds within residues 14,237 to 14,290 of SEQ ID NO: 1899. In one embodiment, the antibody or antigen-binding fragment comprises CDRs of an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 18 / 26. In some embodiments, the antibody or antigen-binding fragment binds within residues 13,935 to 13,947 of SEQ ID NO: 1899. Petition 870260046565, dated 05 / 15 / 2026, p. 26 / 435 In one embodiment, the antibody or antigen-binding fragment comprises CDRs of an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 82 / 858. In some embodiments, the antibody or antigen-binding fragment binds within residues 14,165 to 14,178 of SEQ ID NO: 1899. In one embodiment, the antibody or antigen-binding fragment comprises CDRs of an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 98 / 170.

[0044] In one aspect, the invention provides antibodies or antigen-binding fragments thereof that bind to one or more of the SEA domains of MUC16. In several embodiments, the anti-MUC16 antibodies or antigen-binding fragments bind to any one or more of SEA1, SEA2, SEA3, SEA4, SEA5, SEA6, SEA7, SEA8, SEA9, SEA10, SEA11, SEA12, SEA13, SEA14, SEA15, or SEA16. In one embodiment, the anti-MUC16 antibody or fragment binds within SEA1 (residues 12,074 to 12,229 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA2 (residues 12,230 to 12,387 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA3 (residues 12,388 to 12,543 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA4 (residues 12,544 to 12,698 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA5 (residues 12,699 to 12,699).854 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA6 (residues 12,855 to 13,010 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA7 (residues 13,011 to 13,166 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA8 (residues 13,167 to 13,323 of SEQ ID NO: 1899). In one embodiment. Petition 870260046565, dated 05 / 15 / 2026, p. 27 / 435 In one embodiment, the anti-MUC16 antibody or fragment binds within SEA9 (residues 13,324 to 13,478 of SEQ ID NO: 1899). In another embodiment, the anti-MUC16 antibody or fragment binds within SEA10 (residues 13,479 to 13,634 of SEQ ID NO: 1899). In another embodiment, the anti-MUC16 antibody or fragment binds within SEA11 (residues 13,635 to 13,790 of SEQ ID NO: 1899). In another embodiment, the anti-MUC16 antibody or fragment binds within SEA12 (residues 13,791 to 13,923 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA13 (residues 13,924 to 14,074 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA14 (residues 14,075 to 14,227 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA15 (residues 14,228 to 14,320 of SEQ ID NO: 1899). In one embodiment, the anti-MUC16 antibody or fragment binds within SEA16 (residues 14,321 to 14,322 of SEQ ID NO: 1899).464 of SEQ ID NO: 1899).

[0045] According to another aspect, the present invention provides antibody-drug conjugates comprising an anti-MUC16 antibody or antigen-binding fragment thereof and a therapeutic agent (e.g., a cytotoxic agent). In some embodiments, the antibody or antigen-binding fragment and the cytotoxic agent are covalently linked by a ligand, as discussed in this document. In several embodiments, the anti-MUC16 antibody or antigen-binding fragment may be any of the anti-MUC16 antibodies or fragments described in this document.

[0046] In some embodiments, the cytotoxic agent is selected from an auristatin, a maytansinoid, a tubulisin, a tomaimycin derivative, or a dolastatin derivative. In some cases, the cytotoxic agent is an auristatin selected from Petition 870260046565, dated 05 / 15 / 2026, p. 28 / 435 20 / 202 MMAE or MMAF, or a selected maitansinoid from DM1 or DM4. In some embodiments, the cytotoxic agent is a maytansinoid that has the structure of Formula (I) or Formula (II), as discussed in this document.

[0047] In some forms, the cytotoxic agent is a maytansinoid that has the following structure: ch3

[0048] In some forms, the cytotoxic agent is a maytansinoid that has the following structure:

[0049] In some embodiments, the antibody-drug conjugate comprises an anti-MUC16 antibody or a fragment thereof, and where « is a link to the anti-MUC16 antibody or a fragment thereof.

[0050] In some embodiments, the antibody-drug conjugate Petition 870260046565, dated 05 / 15 / 2026, p. 29 / 435 21 / 202 comprises an anti-MUC16 antibody or a fragment thereof, and where « is a link to the anti-MUC16 antibody or a fragment thereof.

[0051] In some embodiments, the antibody-drug conjugate comprises an anti-MUC16 antibody or a fragment thereof, and where « is a link to the anti-MUC16 antibody or a fragment thereof.

[0052] In some embodiments, the linker comes into contact with the antibody or a fragment thereof by means of a sulfur constituent of a cysteine ​​residue.

[0053] In some embodiments, the antibody-drug conjugate comprises an anti-MUC16 antibody or a fragment thereof, and THE Petition 870260046565, dated 05 / 15 / 2026, p. 30 / 435 22 / 202 a mixture of them, -fem which is a link to the anti-MUC16 antibody or a fragment thereof.

[0054] In some embodiments, the linker comes into contact with the antibody or a fragment thereof by means of a nitrogenous constituent of a lysine residue.

[0055] In any of the various embodiments of antibody-drug conjugates discussed above or in this document, the antibody-drug conjugate may comprise from 1 to 4 cytotoxic agents per anti-MUC16 antibody or fragment thereof.

[0056] According to another aspect, the present invention provides bispecific antigen-binding molecules (e.g., antibodies) that bind to MUC16 and CD3. Such bispecific antigen-binding molecules are also referred to in this document as anti-MUC16 / anti-CD3 bispecific molecules, anti-CD3 / anti-MUC16 bispecific molecules, or MUC16xCD3 bsAbs. The anti-MUC16 portion of the bispecific anti-MUC16 / anti-CD3 molecule is useful for targeting cells (e.g., tumor cells) that express MUC16 (e.g., ovarian tumors), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells.The simultaneous binding of MUC16 to a tumor cell and CD3 to a T cell facilitates the targeted killing (cell lysis) of the targeted tumor cell by the activated T cell. Bispecific anti-T cell molecules... Petition 870260046565, dated 05 / 15 / 2026, page 31 / 435 23 / 202 The MUC16 / anti-CD3 compounds of the invention are therefore useful, among other things, for treating diseases and disorders related to or caused by MUC16-expressing tumors (e.g., ovarian cancers).

[0057] 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 MUC16. The present invention includes bispecific anti-MUC16 / anti-CD3 molecules (e.g., bispecific antibodies) in which each antigen-binding domain comprises a heavy chain variable region (HCVR) paired with a light chain variable region (LCVR). In certain exemplary embodiments of the invention, the anti-CD3 antigen-binding domain and the anti-MUC16 antigen-binding domain each comprise different distinct HCVRs paired with a common LCVR.For example, as illustrated in Example 3 in this document, bispecific antibodies were constructed comprising a first antigen-binding domain that specifically binds CD3, wherein the first antigen-binding domain comprises an HCVR derived from an anti-CD3 antibody paired with an LCVR derived from an anti-MUC16 antibody (e.g., the same LCVR that is included in the anti-MUC16 antigen-binding domain); and a second antigen-binding domain that specifically binds MUC16, wherein the second antigen-binding domain comprises an HCVR / LCVR derived from an anti-MUC16 antibody. In other words, in the exemplary molecules disclosed in this document, the pairing of an HCVR from an anti-CD3 antibody with an LCVR from an anti-MUC16 antibody creates an antigen-binding domain that specifically binds CD3 (but does not bind MUC16). In such embodiments, the first and second domains of... Petition 870260046565, dated 05 / 15 / 2026, p. 32 / 435 24 / 202 antigen-binding molecules comprise distinct anti-CD3 and anti-MUC16 HCVRs, but share a common anti-MUC16 LCVR. In other embodiments, the bispecific antigen-binding molecules comprise distinct anti-CD3 and anti-MUC16 HCVRs, but share a common LCVR. The amino acid sequence of this LCVR is shown, for example, in SEQ ID NO:1890, and the amino acid sequences of the corresponding CDRs (i.e., LCDR1-LCDR2-LCDR3) are shown in SEQ ID NOs:1892, 1894, and 1896, respectively. Genetically modified mice can be used to produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain comprising a variable domain derived from one of two different gene segments of the human light chain variable region.Alternatively, the variable heavy chains can be paired with a common light chain and recombinantly expressed in host cells. Thus, the antibodies of the invention can comprise immunoglobulin heavy chains associated with a single reassigned light chain. In some embodiments, the light chain comprises a variable domain derived from a human Vk1-39 gene segment or a Vk3-20 gene segment. In other embodiments, the light chain comprises a variable domain derived from a human Vk1-39 gene segment reassigned with a human Jk5 or a human Jk1 gene segment.

[0058] The present invention provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the first antigen-binding domain that specifically binds to CD3 comprises any of the amino acid sequences of HCVR, any of the amino acid sequences of LCVR, any of the amino acid sequence pairs of HCVR / LCVR, any of the Petition 870260046565, dated 05 / 15 / 2026, p. 33 / 435 25 / 202 amino acid sequences of heavy chain CDR1-CDR2-CDR3 or any of the light chain CDR1-CDR2-CDR3 amino acid sequences as presented in US publication 2014 / 0088295 published on March 27, 2014 and document PCT / US2016 / 044732 filed on July 29, 2016.

[0059] Furthermore, the present invention provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the first antigen-binding domain that specifically binds to CD3 comprises any of the HCVR amino acid sequences as presented in Tables 16, 18, and 22 hereof. The first antigen-binding domain that specifically binds to CD3 may also comprise any of the LCVR amino acid sequences as presented in Tables 1, 16, 19, and 23 hereof. According to certain embodiments, the first antigen-binding domain that specifically binds to CD3 comprises any of the HCVR / LCVR amino acid sequence pairs as presented in Tables 16, 18, 19, 22, and 23 hereof.The present invention also provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the first antigen-binding domain that specifically binds to CD3 comprises any one of the heavy chain CDR1-CDR2-CDR3 amino acid sequences as presented in Tables 16, 18 and 22 hereof, and / or any one of the light chain CDR1-CDR2-CDR3 amino acid sequences as presented in Tables 1, 16, 19 and 23 hereof.

[0060] According to certain embodiments, the present invention provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the first antigen-binding domain that specifically binds to CD3 comprises a heavy chain variable region (HCVR) having an amino acid sequence as presented in Tables 16, 18 and 22 hereof. Petition 870260046565, dated 05 / 15 / 2026, p. 34 / 435 26 / 202 and 22 in this document or a substantially similar sequence thereof that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0061] The present invention also provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the first antigen-binding domain that specifically binds to CD3 comprises a light chain variable region (LCVR) having an amino acid sequence as presented in Tables 1, 6, 19 and 23 herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0062] The present invention also provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the first antigen-binding domain that specifically binds to CD3 comprises an amino acid sequence pair of HCVR and LCVR (HCVR / LCVR) as presented in Tables 16, 18, 19, 22 and 23 in this document.

[0063] The present invention also provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the first antigen-binding domain that specifically binds to CD3 comprises a heavy chain CDR3 domain (HCDR3) having an amino acid sequence as presented in Tables 16, 18 and 22 herein, or a substantially similar sequence thereto 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 as presented in Tables 1, 16, 19, and 23 herein, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. Petition 870260046565, dated 05 / 15 / 2026, p. 35 / 435 27 / 202

[0064] In certain embodiments, the first antigen-binding domain that specifically binds to CD3 comprises an HCDR3 / LCDR3 amino acid sequence pair as shown in Tables 16, 18, 19, 22 and 23 in this document.

[0065] The present invention also provides bispecific antigen-binding molecules anti-CD3 / anti-MUC16, wherein the first antigen-binding domain that specifically binds to CD3 comprises a heavy chain CDR1 domain (HCDR1) having an amino acid as presented in Tables 16, 18 and 22 herein, or a substantially similar sequence thereof 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 as presented in Tables 16, 18 and 22, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity;a heavy chain CDR3 (HCDR3) domain having an amino acid as shown in Tables 16, 18 and 22, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence as shown in Tables 1, 16, 19 and 23 in this document, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a CDR2 light chain domain (LCDR2) having an amino acid sequence as presented in Tables 1, 16, 19 and 23 in this document, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity, and a CDR3 light chain domain (LCDR3) having; Petition 870260046565, dated 05 / 15 / 2026, p. 36 / 435 28 / 202 an amino acid sequence as presented in Tables 1, 16, 19 and 23 in this document, or a substantially similar sequence that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0066] Certain exemplary, non-limiting anti-CD3 / anti-MUC16 bispecific antigen-binding molecules of the invention include a first antigen-binding domain that specifically binds to CD3 comprising HCDR1-HCDR2-HCDR3LCDR1-LCDR2-LCDR3 domains, respectively, which have the amino acid sequences as presented in Tables 16, 18, 19, 22 and 23 in this document.

[0067] The present invention further provides a bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to human CD3 comprises heavy chain complementarity-determining regions (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) comprising an amino acid sequence as presented in Table 16, Table 18, or Table 22 and light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) of a light chain variable region (LCVR) comprising an amino acid sequence as presented in Table 1, Table 16, Table 19 or Table 23.

[0068] In another aspect, the invention provides a bispecific antigen-binding molecule in which the first antigen-binding domain that specifically binds to human CD3 comprises heavy chain complementarity-determining regions (HCDR1, HCDR2 and HCDR3) of a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 1730, 1762, 1778, 1786 and 1866, and light chain complementarity-determining regions (LCDR1, LCDR2 and LCDR3) from Petition 870260046565, dated 05 / 15 / 2026, page 37 / 435 29 / 202 of a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 26.

[0069] The invention further provides a bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to human CD3 comprises three heavy chain complementarity-determining regions (A1HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity-determining regions (A1-LCDR1, A1-LCDR2 and A1LCDR3), wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788 and 1868; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734, 1766, 1782, 1790 and 1870; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1736, 1768, 1784, 1792 and 1872; A1-LCDR1 comprises an amino acid sequence with SEQ ID NO: 28; A1-LCDR2 comprises an amino acid sequence with SEQ ID NO: 30;and A1-LCDR3 comprises an amino acid sequence with SEQ ID NO: 32.;

[0070] In a further aspect, the invention provides a bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to human CD3 comprises the heavy and light chain CDRs of a pair of HCVR / LCVR amino acid sequences selected from the group consisting of: SEQ ID NOs: 1730 / 26, 1762 / 26, 1778 / 26, 1786 / 26 and 1866 / 26

[0071] In another aspect, the invention provides a bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to human CD3 comprises three heavy chain complementarity-determining regions. Petition 870260046565, dated 05 / 15 / 2026, p. 38 / 435 30 / 202 (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity-determining regions (A1-LCDR1, A1-LCDR2 and A1LCDR3), and wherein the second antigen-binding domain that specifically binds to human MUC16 comprises three heavy chain complementarity-determining regions (A2-HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity-determining regions (A2-LCDR1, A2-LCDR2 and A2LCDR3); wherein A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788 and 1868; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734, 1766, 1782, 1790 and 1870; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1736, 1768, 1784, 1792 and 1872; A1-LCDR1 comprises an amino acid sequence with SEQ ID NO: 28; A1-LCDR2 comprises an amino acid sequence with SEQ ID NO: 30;where A1-LCDR3 comprises an amino acid sequence with SEQ ID NO: 32; and where A2-HCDR1 comprises an amino acid sequence with SEQ ID NO: 20; A2-HCDR2 comprises an amino acid sequence with SEQ ID NO: 22; A2-HCDR3 comprises an amino acid sequence with SEQ ID NO: 24; A2-LCDR1 comprises an amino acid sequence with SEQ ID NO: 28; A2LCDR2 comprises an amino acid sequence with SEQ ID NO: 30; and A2-LCDR3 comprises an amino acid sequence with SEQ ID NO: 32.

[0072] Certain exemplary, non-limiting anti-CD3 / anti-MUC16 bispecific antigen-binding molecules of the invention include a first antigen-binding domain that specifically binds to CD3 comprising a heavy chain comprising variable domain scaffold regions having a sequence of Petition 870260046565, dated 05 / 15 / 2026, p. 39 / 435 31 / 202 amino acids selected from FR1 (SEQ ID NO: 1903), FR2 (SEQ ID NO: 1904), FR3 (SEQ ID NO: 1905) and FR4 (SEQ ID NO: 1906).

[0073] In further embodiments, exemplary anti-CD3 / anti-MUC16 bispecific antigen-binding molecules of the invention include a bispecific antigen-binding molecule in which the first antigen-binding domain that specifically binds to human CD3 comprises an HCVR comprising HCDR1-HCDR2-HCDR3 having the amino acid sequences SEQ ID NOs: 1907-1908-1909.

[0074] The present invention also provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the second antigen-binding domain that specifically binds to MUC16 comprises a heavy chain variable region (HCVR) having the amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 202, 218, 234, 250, 258, 274, 282, 298, 314, 330, 346, 362 and 378, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0075] The present invention also provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the second antigen-binding domain that specifically binds to MUC16 comprises a light chain variable region (LCVR) having the amino acid sequence selected from the group consisting of SEQ ID NOs: 10; 26; 42; 58; 74; 90; 106; 122; 138; 154; 170; 186; 210; 226; 242; 266; 290; 306; 322; 338; 354; 370; 386; 1936; and 394, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0076] The present invention also provides molecules Petition 870260046565, dated 05 / 15 / 2026, p. 40 / 435 32 / 202 bispecific anti-CD3 / anti-MUC16, wherein the second antigen-binding domain that specifically binds to MUC16 comprises an amino acid sequence pair of HCVR and LCVR (HCVR / LCVR) selected from the group consisting of SEQ ID NOs:18 / 26.

[0077] The present invention also provides bispecific anti-CD3 / anti-MUC16 molecules, wherein the second antigen-binding domain that specifically binds to MUC16 comprises a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 208, 224, 240, 256, 264, 280, 288, 304, 320, 336, 352, 368 and 384, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98% or at least 99% sequence identity;and a CDR3 light chain domain (LCDR3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 216, 232, 248, 272, 296, 312, 328, 344, 360, 376, 392, 400 and 1942, or a substantially similar sequence thereof that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0078] In certain embodiments, the second antigen-binding domain that specifically binds to MUC16 comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 24 / 32.

[0079] The present invention also provides bispecific antigen-binding molecules anti-CD3 / anti-MUC16, wherein the second antigen-binding domain that specifically binds to MUC16 comprises a heavy chain CDR1 domain (HCDR1) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, Petition 870260046565, dated 05 / 15 / 2026, p. 41 / 435 33 / 202 180, 196, 204, 220, 236, 252, 260, 276, 284, 300, 316, 332, 348, 364 and 380, or a substantially similar sequence thereof that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 206, 222, 238, 254, 262, 278, 286, 302, 318, 334, 350, 366 and 382, ​​or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity;a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 208, 224, 240, 256, 264, 280, 304, 320, 336, 352, 368 and 384, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 396, 212, 228, 244, 396, 268, 396, 292, 308, 324, 340, 356, 372, 1938 and 388, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity;and a CDR2 light chain domain (LCDR2) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 398, 214, 230, 246, 398, 270, 398, 294, 310, 326, 342, 358, 374, 1940 and 390, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a CDR3 light chain domain (LCDR3) having an amino acid sequence; Petition 870260046565, dated 05 / 15 / 2026, p. 42 / 435 34 / 202 selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 400, 216, 232, 248, 400, 272, 400, 296, 312, 328, 344, 360, 376, 1942 and 392, or a substantially similar sequence thereof that has at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0080] Certain exemplary, non-limiting anti-CD3 / anti-MUC16 bispecific antigen-binding molecules of the invention include a second antigen-binding domain that specifically binds to MUC16 comprising HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, respectively, having amino acid sequences selected from the group consisting of: SEQ ID NOs: 20-22-24-28-30-32.

[0081] In a related embodiment, the invention includes bispecific anti-CD3 / anti-MUC16 antigen-binding molecules wherein the second antigen-binding domain that specifically binds to MUC16 comprises heavy and light chain CDR domains contained within heavy and light chain variable region (HCVR / LCVR) sequences selected from the group consisting of SEQ ID NOs: 18 / 26.

[0082] In one embodiment, the invention provides a bispecific anti-CD3 / anti-MUC16 antibody comprising an anti-MUC16 binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1959 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960, and an anti-CD3 binding arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1961 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960. In another embodiment, the invention provides a bispecific anti-CD3 / anti-MUC16 antibody comprising an arm Petition 870260046565, dated 05 / 15 / 2026, pp. 43 / 435 35 / 202 anti-MUC16 linker comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1959 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960, and an anti-CD3 linker arm comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1962 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960.

[0083] In another aspect, the invention provides a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to human CD3 and a second antigen-binding domain that binds to human MUC16, wherein the second antigen-binding domain is derived from the antibody or antigen-binding fragment of any of the anti-MUC16 antibodies of the invention. In a further aspect, the invention provides a bispecific antigen-binding molecule comprising a first antigen-binding domain that binds specifically to human CD3, and a second antigen-binding domain that binds specifically to human MUC16.

[0084] The invention further provides a bispecific antigen-binding molecule that binds to human cells expressing human CD3 and to cynomolgus monkey cells expressing cynomolgus CD3. In another aspect, the bispecific antigen-binding molecule binds to human cells expressing human MUC16.

[0085] In another aspect, the invention provides a bispecific antigen-binding molecule that inhibits tumor growth in immunocompromised mice bearing human ovarian cancer xenografts. The invention further provides a bispecific antigen-binding molecule that suppresses tumor growth of established tumors in mice. Petition 870260046565, dated 05 / 15 / 2026, p. 44 / 435 36 / 202 immunocompromised patients carrying human ovarian cancer xenografts.

[0086] In another aspect, the invention provides a bispecific antigen-binding molecule comprising i) a first antigen-binding domain that specifically binds an effector cell with an EC50 value greater than about 4 nM, and ii) a second antigen-binding domain that specifically binds a human ovarian tumor target cell with an EC50 value less than 3 nM, wherein such EC50 binding value is measured in an in vitro FACS binding assay.

[0087] In one embodiment, the bispecific antigen-binding molecule may include a second antigen-binding domain that specifically binds to ovarian tumor target cells with an EC50 value less than about 2 nM. In some cases, the first antigen-binding domain specifically binds to each of human CD3 and cynomolgus CD3 with an EC50 value greater than about 40 nM, greater than about 100 nM, greater than about 200 nM, greater than about 300 nM, greater than about 400 nM, greater than about 500 nM, or greater than about 1 pM. In some cases, the first antigen-binding domain specifically binds to each of human CD3 and cynomolgus CD3 with weak or unmeasurable binding affinity.

[0088] In some embodiments, the antigen-binding molecule induces T-cell-mediated tumor cell death with an EC50 value lower than about 31 pM, as measured in an in vitro T-cell-mediated tumor cell death assay, for example, where the tumor cells are OVCAR3 cells.

[0089] In some applications, the first antigen-binding domain binds to human CD3 with a Kd value greater than about 11 nM, as measured in a resonance binding assay of Petition 870260046565, dated 05 / 15 / 2026, p. 45 / 435 37 / 202 surface plasmon in vitro. In some cases, the first antigen-binding domain binds to each of human CD3 and cynomolgus CD3 with a Kd value greater than about 15 nM, greater than about 30 nM, greater than about 60 nM, greater than about 120 nM, greater than about 300 nM, or greater than about 500 nM, as measured in an in vitro surface plasmon resonance binding assay.

[0090] In certain embodiments, the anti-CD3 antibodies of the invention, antigen-binding fragments and bispecific antibodies thereof were produced by replacing the amino acid residues of a parent in a stepwise manner based on differences between the germline sequence and the parental antibody sequence.

[0091] In some embodiments, the invention provides a bispecific antigen-binding molecule, wherein the second antigen-binding domain competes for binding to human MUC16 with a reference antigen-binding protein comprising three heavy chain complementarity-determining regions (A2HCDR1, A2-HCDR2 and A2-HCDR3) and three light chain complementarity-determining regions (A2-LCDR1, A2-LCDR2 and A2LCDR3), wherein A2-HCDR1 comprises an amino acid sequence of SEQ ID NO: 20; A2-HCDR2 comprises an amino acid sequence of SEQ ID NO: 22; A2-HCDR3 comprises an amino acid sequence of SEQ ID NO: 24; A2-LCDR1 comprises an amino acid sequence of SEQ ID NO: 28; A2LCDR2 comprises an amino acid sequence of SEQ ID NO: 30; and A2-LCDR3 comprises an amino acid sequence of SEQ ID NO: 32.In some embodiments, the invention provides a bispecific antigen-binding molecule, wherein the second antigen-binding domain competes for binding to human MUC16 with a... Petition 870260046565, dated 05 / 15 / 2026, page 46 / 435 38 / 202 reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence of SEQ ID NO: 18, and a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 26.

[0092] In some embodiments, the invention provides a bispecific antigen-binding molecule, in which the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising three heavy chain complementarity-determining regions (A1HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity-determining regions (A1-LCDR1, A1-LCDR2 and A1LCDR3), A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788 and 1868; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734, 1766, 1782, 1790 and 1870; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1736, 1768, 1784, 1792 and 1872; A1-LCDR1 comprises an amino acid sequence with SEQ ID NO: 28; A1LCDR2 comprises an amino acid sequence with SEQ ID NO: 30;and A1-LCDR3 comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, the invention provides the bispecific antigen-binding molecule, wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1730, 1762, 1778, 1786 and 1866, and a light chain variable region (LCVR) comprising an amino acid sequence of SEQ; Petition 870260046565, dated 05 / 15 / 2026, page 47 / 435 39 / 202 ID NO: 26.

[0093] In some embodiments, the invention provides a bispecific antigen-binding molecule, wherein the first antigen-binding domain competes for binding to human CD3 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1730, 1762, 1778, 1786 and 1866, and a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 26; and wherein the second antigen-binding domain competes for binding to human MUC16 with a reference antigen-binding protein comprising a heavy chain variable region (HCVR) comprising an amino acid sequence of SEQ ID NO: 18, and a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 26.

[0094] In one aspect, the invention provides a pharmaceutical composition comprising an anti-MUC16 antigen-binding molecule or a bispecific anti-MUC16 / anti-CD3 antigen-binding molecule and a pharmaceutically acceptable diluent or vehicle. The invention further provides a method for treating cancer in an individual, wherein the method comprises administering to the individual the pharmaceutical composition comprising an anti-MUC16 antigen-binding molecule or a bispecific anti-MUC16 / anti-CD3 antigen-binding molecule and a pharmaceutically acceptable diluent or vehicle. In some embodiments, the cancer is selected from the group consisting of cancers that include ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma mass-forming type, cervical adenocarcinoma and Petition 870260046565, dated 05 / 15 / 2026, page 48 / 435 40 / 202 adenocarcinoma of the gastric tract. In some cases, the cancer is ovarian cancer.

[0095] In another aspect, the present invention provides nucleic acid molecules encoding any of the HCVR, LCVR, or CDR sequences of the anti-CD3 / anti-MUC16 bispecific antigen-binding molecules disclosed herein, including nucleic acid molecules comprising the polynucleotide sequences as presented in Tables 2, 17, 20, 21, 23, and 25 herein, as well as nucleic acid molecules comprising two or more of the polynucleotide sequences as presented in Tables 2, 17, 20, 21, 23, and 25 in any functional combination or arrangement thereof. The invention also encompasses 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 conditions that permit antibody production, and recovering the antibodies produced.

[0096] The present invention includes bispecific anti-CD3 / anti-MUC16 antigen-binding molecules in which any of the aforementioned antigen-binding domains that specifically bind to CD3 are combined, connected, or otherwise associated with any of the aforementioned antigen-binding domains that specifically bind to MUC16 to form a bispecific antigen-binding molecule that binds to both CD3 and MUC16.

[0097] The present invention includes bispecific antigen-binding molecules anti-CD3 / anti-MUC16 having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or an antibody Petition 870260046565, dated 05 / 15 / 2026, p. 49 / 435 41 / 202 devoid of a fucose chemical moiety present in the oligosaccharide chain, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, a glycosylation modification can be produced in order to modify complement-dependent cytotoxicity (CDC).

[0098] In another aspect, the invention provides a pharmaceutical composition comprising a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule as disclosed herein and a pharmaceutically acceptable carrier. In a related aspect, the invention presents a composition that is a combination of a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule. Exemplary agents that can be advantageously combined with a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule are discussed in detail elsewhere in this document.

[0099] In yet another aspect, the invention provides therapeutic methods for targeting / killing tumor cells expressing MUC16 using a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule of the invention to an individual in need thereof.

[00100] The present invention also includes the use of a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule of the invention in the manufacture of a medicament for the treatment of a disease or Petition 870260046565, dated 05 / 15 / 2026, p. 50 / 435 42 / 202 disorder related to or caused by cells expressing MUC16.

[00101] In another aspect, the present invention provides a method for detecting MUC16 in a biological sample comprising: obtaining a biological sample from an individual and detecting whether MUC16 is present in the biological sample by contacting the biological sample with an anti-MUC16 antibody or antigen-binding fragment thereof and detecting the binding between MUC16 and the anti-MUC16 antibody or antigen-binding fragment. In some cases, the biological sample is a tissue or fluid sample selected from plasma, serum, ascites, ovary, uterus, cervix, liver, bladder, pancreas, stomach, small and large intestines, gallbladder, breast, lung, kidney, salivary and lacrimal glands or any epithelioid malignancy thereof.In some embodiments, the antibody or antigen-binding fragment binds to human MUC16 within one or more of five proximal membrane SEA domains of human MUC16 corresponding to residues 13,791 to 14,451 of SEQ ID NO: 1899. In some embodiments, the antibody or antigen-binding fragment binds to human MUC16 within residues 13,810 to 14,451 of SEQ ID NO: 1899. In some embodiments, the antibody or antigen-binding fragment binds to any one or more of SEA1, SEA2, SEA3, SEA4, SEA5, SEA6, SEA7, SEA8, SEA9, SEA10, SEA11, SEA12, SEA13, SEA14, SEA15, or SEA16 of human MUC16.

[00102] In another aspect, the invention provides a method for detecting MUC16 in a patient comprising: obtaining a tissue sample from the patient; and detecting whether MUC16 is present in the tissue sample by contacting the tissue sample with an anti-MUC16 antibody and detecting the binding between MUC16 and the anti-MUC16 antibody. In some cases, the method further comprises diagnosing the patient with cancer when MUC16 is present. Petition 870260046565, dated 05 / 15 / 2026, page 51 / 435 43 / 202 in the tissue sample is detected. In one embodiment, the tissue sample is ovarian tissue. In some cases, the anti-MUC16 antibody is specific for an epitope within residues 12,783 to 13,467 of SEQ ID NO: 1899. In one embodiment, the anti-MUC16 antibody comprises CDRs of an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 202 / 210. In some cases, the anti-MUC16 antibody is specific for an epitope with residues 13,810 to 14,451 of SEQ ID NO: 1899. In one embodiment, the anti-MUC16 antibody comprises CDRs of a pair of amino acid sequences of HCVR / LCVR selected from the group consisting of SEQ ID NO: 250 / 1936, 258 / 266, 314 / 322 and 1944 / 1952.

[00103] In another aspect, the invention provides a method for detecting MUC16 in a patient comprising: obtaining a plasma sample from the patient; and detecting whether MUC16 is present in the plasma sample by contacting the plasma sample with an anti-MUC16 antibody comprising CDRs of an HCVR / LCVR pair comprising the amino acid sequences of SEQ ID NO: 202 / 210, and detecting the binding between MUC16 and the anti-MUC16 antibody. In some cases, the method further comprises diagnosing the patient with cancer when the presence of MUC16 in the plasma sample is detected. In some embodiments, the method further comprises administering an effective amount of a bispecific anti-CD3xMUC16 antibody to the diagnosed patient. In some embodiments, the method further comprises administering an effective amount of an ADC comprising an anti-MUC16 antibody or antigen-binding fragment thereof and a cytotoxic agent to the diagnosed patient.

[00104] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to MUC16, wherein the antibody or antigen-binding fragment comprises Petition 870260046565, dated 05 / 15 / 2026, p. 52 / 435 44 / 202 CDRs of a pair of amino acid sequences HCVR / LCVR selected from the group consisting of SEQ ID NOs: 202 / 210, 250 / 1936, 258 / 266, 314 / 322, 82 / 858, 98 / 170 and 1944 / 1952. In some embodiments, the antibody or antigen-binding fragment thereof comprises domains of HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3, respectively, selected from the group consisting of SEQ ID NOs: 204-206-208-212-214-216; 252-254-256-1938-1940-1942; 260-262-264-268-270-272; 316-318-320-324-326-328; 84-86-88-1892-1894-1896; 100-102-104-172-174-176; and 1946-1948-1950-1954-1956-1958. In some embodiments, the antibody or antigen-binding fragment comprises a pair of HCVR / LCVR amino acid sequences selected from the group consisting of 202 / 210, 250 / 1936, 258 / 266, 314 / 322, 82 / 858, 98 / 170 and 1944 / 1952.

[00105] Other modalities will become evident from a review of the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[00106] Figures 1, 2 and 3 illustrate pharmacokinetic profiles of bispecific anti-MUC16 x CD3 antibodies in wild-type mice (Figure 1), humanized CD3 mice (Figure 2) or humanized MUC16 x CD3 mice (Figure 3).

[00107] Figure 4 shows the results of model study 1. OVCAR-3 (Mean Radiance [p / s / cm2 / sr] on Day 6). All groups had similar tumor burden as assessed by BLI before dosing was initiated. Data shown are tumor burden as assessed by BLI on Day 6 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t-tests. There was no significant difference in tumor burden between the groups.

[00108] Figure 5 shows the results of model study 1. Petition 870260046565, dated 05 / 15 / 2026, p. 53 / 435 45 / 202 OVCAR-3 (Mean Radiance [p / s / cm22 / sr] on Day 20). BSMUC16 / CD3001 significantly reduces tumor burden at 0.1 and 0.5 mg / kg. Human T-cell-grafted NSG mice were implanted with human OVCAR-3 / Luc cells. Treatment began 6 days after tumor implantation. Mice were treated on Days 6, 10, 13, 16, and 21 with 0.01, 0.1, or 0.5 mg / kg of BSMUC16 / CD3001 administered IP or treated with a CD3-binding control or a non-binding control (0.5 mg / kg IP). Data shown are tumor burden as assessed by BLI on Day 20 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t-tests. Treatment with BSMUC16 / CD3-001 was compared with a non-binding control (** p < 0.01 for 0.5 mg / kg, # p < 0.05 for 0.1 mg / kg of BSMUC16 / CD3-001).

[00109] Figure 6 shows the results of the OVCAR-3 model study 1 (Multiplication alteration in evident BLI tumors between D6 and D20). BSMUC16 / CD3-001 significantly reduces the multiplication alteration in tumor burden at 0.01, 0.1, and 0.5 mg / kg. Human T-cell-grafted NSG mice were implanted with human OVCAR-3 / Luc cells. Mice were treated on Days 6, 10, 13, 16, and 21 with 0.01, 0.1, or 0.5 mg / kg of BSMUC16 / CD3-001 administered IP or treated with a CD3-binding control or a non-binding control (0.5 mg / kg IP). The data shown are the multiplication alteration in tumor burden from the first measurement (taken before treatment was initiated) and at Day 20 at the end of the study. Statistical significance was determined using unpaired non-parametric Mann-Whitney t-tests.Treatment with BSMUC16 / CD3-001 was compared with the non-binding control (** p < 0.01 for 0.5 mg / kg, # p < 0.05 for 0.1 mg / kg, $ p < 0.05 for 0.01 mg / kg of BSMUC16 / CD3-001). Petition 870260046565, dated 05 / 15 / 2026, p. 54 / 435 46 / 202

[00110] Figure 7 shows the results of model study 2. OVCAR-3 (Mean Radiance [p / s / cm2 / sr] on Day 4). All groups had similar tumor burden as assessed by BLI before dosing was initiated. The data shown are the tumor burden as assessed by BLI on Day 4 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t-tests. There was no significant difference in tumor burden on Day 4 between the groups.

[00111] Figure 8 shows the results of the OVCAR-3 model study 2 (Mean Radiance [p / s / cm22 / sr] on Day 25). BSMUC16 / CD3005 significantly reduces tumor burden at 0.5, 1, and 5 mg / kg. Human T-cell-grafted NSG mice were implanted with human OVCAR-3 / Luc cells. Treatments began 5 days after tumor implantation. Mice were treated on Days 5, 8, 12, 15, 19, and 22 with 0.1, 0.5, 1, or 5 mg / kg of REGN4019 administered IV or a CD3-binding control or a non-binding control (5 mg / kg IV). Data shown are tumor burden as assessed by BLI on Day 25 after tumor implantation. Statistical significance was determined using unpaired nonparametric Mann-Whitney t-tests. Treatment with BSMUC16 / CD3-005 was compared with the non-binding control (** p < 0.01 for 5 mg / kg, ## p < 0.01 for 1 mg / kg, $$ p < 0.01 for 0.5 mg / kg of BSMUC16 / CD3-005).

[00112] Figure 9 shows the results of study 2 of the OVCAR-3 model (Multiplication alteration in evident BLI tumors between D4 and D25). BSMUC16 / CD3-005 significantly reduces tumor growth at 0.5, 1, and 5 mg / kg. Human T-cell-grafted NSG mice were implanted with human OVCAR-3 / Luc cells. Mice were treated on Days 5, 8, 12, 15, 19, and 22 with 0.1, 0.5, 1, or 5 mg / kg of REGN4019. Petition 870260046565, dated 05 / 15 / 2026, p. 55 / 435 47 / 202 administered IV or treated with a CD3-binding control or a non-binding control (5 mg / kg IV). The data shown are the change in tumor burden multiplication from the first measurement (taken the day before treatment began) and at Day 25, at the end of the study. Statistical significance was determined using unpaired non-parametric Mann-Whitney t-tests. Treatment with BSMUC16 / CD3-005 was compared to the non-binding control (** p < 0.01 for 5 mg / kg, ## p < 0.01 for 1 mg / kg, $$ p < 0.01 for 0.5 mg / kg of REGN4019).

[00113] Figure 10 shows the results of the ID8VEGF / huMUC16 model. Tumor size on Day 47 after BSMUC16 / CD3-001 implantation significantly reduces tumor growth in a syngeneic model when treatment begins on the day of implantation or 10 days after tumor implantation. Mice expressing human CD3 in place of mouse CD3 and a chimeric MUC16 molecule were implanted with a murine ovarian tumor cell line expressing a human MUC16 moiety. Mice were administered BSMUC16 / CD3-001 (100 µg IP) on the day of implantation or 10 days after implantation, or administered with a CD3-binding control (100 µg IP) on the day of implantation. Mice were treated on Days 0, 4, 7, 10, 13, 17, 20, or 24 for the immediate treatment group and on Days 10, 13, 17, 20, and 24 for the group in which dosing began on D10. The data shown are the tumor volume on Day 47 after implantation.Statistical significance was determined using unpaired nonparametric Mann-Whitney t-tests. Treatment with BSMUC16 / CD3-001 was compared with the CD3 binding control (** p < 0.01 initiated on D0, * p < 0.05 initiated on D10).

[00114] Figures 11A to 11C illustrate the results of flow cytometry (or FACS) analysis of bispecific antibody binding. Petition 870260046565, dated 05 / 15 / 2026, p. 56 / 435 48 / 202 selected for PEO-1, OVCAR3-Luc, Jurkat cells and cynomolgus T cells. Titration analysis was performed by testing a range of serial dilutions of each antibody: bispecific MUC16xCD3 antibodies BSMUC16 / CD3-001, BSMUC16 / CD3-002 or BSMUC16 / CD3-003 or a first or second isotype control antibody (which does not cross-react with CD3 or MUC16).

[00115] Figures 12A and 12B represent examples of cell extermination of PEO-1 (Figure 12A) or OVCAR3-Luc (Figure 12B) in a cytotoxicity assay 48 hours after antiMUC16xanti-CD3 treatment in the presence of human PBMCs. DETAILED DESCRIPTION

[00116] Before the present invention is described, it should be understood that the present invention is not limited to the particular experimental methods and conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[00117] Unless otherwise defined, all technical and specific terms used in this document have the same meaning that is commonly understood by a common skill element in the art to which the present invention pertains. As used herein, the term about, when used in reference to a particular recited numerical value, means that the value may vary from the stated value by no more than 1%. For example, as used in this document, the expression about 100 includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[00118] Although any methods and materials similar or equivalent to those described in this document may be Petition 870260046565, dated 05 / 15 / 2026, p. 57 / 435 49 / 202 used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned in this specific report are incorporated herein by reference in their entirety. DEFINITIONS

[00119] The term CD3, as used in this document, refers to an antigen that is expressed on T cells as part of the multimolecular T cell receptor (TCR) and that consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acid sequence as presented in SEQ ID NO: 1897; human CD3-delta comprises the amino acid sequence as presented in SEQ ID NO: 1898. All references to proteins, polypeptides, and protein fragments in this document are intended to refer to the human version of the respective protein, polypeptide, or protein fragment except where explicitly specified as being from a non-human species. Therefore, the term CD3 means human CD3, except where specified as being from a non-human species, for example, mouse CD3, monkey CD3, etc.

[00120] As used in this document, a CD3-binding antibody or an anti-CD3 antibody includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (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 (e.g., CD3 dimers gamma / epsilon, delta / epsilon, and zeta / zeta). The antibodies and antigen-binding fragments Petition 870260046565, dated 05 / 15 / 2026, p. 58 / 435 50 / 202 antigens of the present invention can bind to soluble CD3 and / or CD3 expressed on cell surfaces. Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants such as, for example, monomeric and dimeric CD3 constructs, lacking a transmembrane domain or otherwise not associated with a cell membrane.

[00121] As used in this document, the term cell-surface-expressed CD3 means one or more CD3 proteins that are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. Cell-surface-expressed CD3 includes CD3 proteins contained within the context of a functional T-cell receptor on a cell membrane. The term cell-surface-expressed CD3 includes the CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The term cell-surface-expressed CD3 also includes a CD3 chain (e.g., CD3-epsilon, CD3-delta, or CD3-gamma) that is expressed alone, without other CD3 chain types, on the surface of a cell.A cell surface-expressed CD3 may comprise or consist of a CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, a 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 modified to express CD3 on its surface.

[00122] The expression MUC16, as used in this document, refers to mucin 16. MUC16 is a membrane glycoprotein. Petition 870260046565, dated 05 / 15 / 2026, page 59 / 435 51 / 202 is a highly glycosylated integral single-domain transmembrane protein that is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is presented in SEQ ID NO:1899.

[00123] As used in this document, an antibody that binds to MUC16 or an anti-MUC16 antibody includes antibodies and antigen-binding fragments thereof that specifically recognize MUC16.

[00124] The term antigen-binding molecule includes antibodies and antibody antigen-binding fragments, including, for example, bispecific antibodies.

[00125] The term antibody, as used in this document, means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., MUC16 or CD3). The term antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a variable heavy chain region (abbreviated in this document as HCVR or VH) and a constant heavy chain region. The constant heavy chain region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a variable light chain region (abbreviated in this document as LCVR or VL) and a constant light chain region.The constant light chain region comprises a domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). Each VH and VL is... Petition 870260046565, dated 05 / 15 / 2026, p. 60 / 435 52 / 202 is composed of three CDRs and four FRs, arranged from amino-terminal to carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-MUC16 antibody or anti-CD3 antibody (or the antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. A consensus amino acid sequence may be defined based on a side-by-side analysis of two or more CDRs.

[00126] The term antibody, as used in this document, also includes antigen-binding fragments of complete antibody molecules. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like, as used in this document, include any naturally occurring, enzymatically obtainable, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, for example, from complete antibody molecules using any standard techniques such as proteolytic digestion or recombinant genetic modification techniques involving the manipulation and expression of DNA encoding antibody variables and optionally constant domains.Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or it can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids. Petition 870260046565, dated 05 / 15 / 2026, p. 61 / 435 53 / 202 etc.

[00127] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constricted FR3-CDR3-FR4 peptide. Other modified molecules, such as domain-specific antibodies, single-domain antibodies, domain-related antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.).Small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains are also encompassed within the antigen-binding fragment expression, as used in this document.

[00128] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR that is adjacent to or framed with one or more scaffold sequences. In antigen-binding fragments that have a VH domain associated with a VL domain, the VH and VL domains may be situated relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[00129] In certain modalities, a link fragment to Petition 870260046565, dated 05 / 15 / 2026, p. 62 / 435 54 / 202 An antibody antigen may contain at least one variable domain covalently linked to at least one constant domain. Illustrative, non-limiting configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) Vh-Ch1; (ii) Vh-Ch2; (iii) Vh-Ch3; (iv) Vh-Ch1-Ch2; (v) Vh-Ch1-Ch2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) VlCh1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) Vl-Ch2-Ch3; and (xiv) Vl-Cl. In any configuration of variable and constant domains, including any of the example configurations listed above, the variable and constant domains can be directly linked to each other or can be linked by a full or partial articulation or linking region.A linking region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, 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 listed above in non-covalent association with each other and / or with one or more monomeric Vh or Vl domains (e.g., by disulfide linkage(s)).

[00130] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, where each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including antibody formats Petition 870260046565, dated 05 / 15 / 2026, page 63 / 435 55 / 202 exemplary bispecific antibody disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.

[00131] The antibodies of the present invention can function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). 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 reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize the antibody bound to a target cell and thus lead to lysis of the target cell. CDC and ADCC can be measured using assays that are well-known and available in the art. (See, for example, U.S. Patents 5,500,362 and 5,821,337 and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656).The constant region of an antibody is important in its ability to fix complement and mediate cell-dependent cytotoxicity. Therefore, the isotype of an antibody can be selected based on the likelihood that the antibody will mediate cytotoxicity.

[00132] In certain embodiments of the invention, the monospecific anti-MUC16 antibodies or bispecific anti-MUC16 / anti-CD3 antibodies of the invention are human antibodies. The term human antibodies, as used herein, is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include residues Petition 870260046565, dated 05 / 15 / 2026, page 64 / 435 56 / 202 of amino acids not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by site-specific or random mutagenesis in vitro or by somatic mutation in vivo), for example, in CDRs and, in particular, CDR3. However, the term human antibodies, as used in this document, is not intended to include antibodies in which germline-derived CDR sequences from another mammalian species, such as a mouse, have been grafted onto human scaffold sequences.

[00133] The antibodies of the invention may, in some embodiments, be recombinant human antibodies. The term recombinant human antibodies, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using 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., a mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6,287 to 6,295) or antibodies prepared, expressed, created or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences.These recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal for human Ig sequences is used, in vivo somatic mutagenesis) and, thus, the amino acid sequences of the regions are altered. Petition 870260046565, dated 05 / 15 / 2026, page 65 / 435 57 / 202 of recombinant antibody Vh and Vl are sequences that, although derived from and related to human germline Vh and Vl sequences, may not naturally exist within the human germline antibody repertoire in vivo.

[00134] Human antibodies can exist in two forms that are associated with articulation heterogeneity. In one form, an immunoglobulin molecule comprises a stable construct of four chains of approximately 150 to 160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked by interchain disulfide bonds and a molecule of about 75 to 80 kDa is formed composed of a covalently coupled light and heavy chain (half antibody). These forms have been extremely difficult to separate, even after affinity purification.

[00135] The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody's binding region isotype. A single amino acid substitution in the binding region of human IgG4 can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) at levels typically observed with the use of a human IgG1 joint. The present invention encompasses antibodies that have one or more mutations in the joint, Ch2 or Ch3 region that may be desirable, for example, in production, to enhance the yield of the desired antibody form.

[00136] The antibodies of the invention may be isolated antibodies. An isolated antibody, as used herein, means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, a Petition 870260046565, dated 05 / 15 / 2026, p. 66 / 435 58 / 202 An antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an isolated antibody for the purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemical agents.

[00137] The present invention also includes single-arm antibodies that bind to MUC16. As used herein, a single-arm antibody means an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The single-arm antibodies of the present invention may comprise any of the HCVR / LCVR or CDR amino acid sequences as presented in Table 1.

[00138] The anti-MUC16 or anti-MUC16 / anti-CD3 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the scaffold and / or CDR regions of the variable heavy and light chain domains compared to the corresponding germline sequences from which the antibodies were derived.Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any one of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more. Petition 870260046565, dated 05 / 15 / 2026, p. 67 / 435 59 / 202 scaffold and / or CDR regions are mutated for the corresponding residue (or residues) of the germline sequence from which the antibody was derived, or for the corresponding residue (or residues) of another human germline sequence, or for a conservative amino acid substitution of the corresponding germline residue (or residues) (such sequence alterations are referred to herein collectively as germline mutations). A person of ordinary skill in the art, beginning with the variable heavy and light chain region sequences disclosed herein, can readily produce numerous antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof.In certain embodiments, all of the scaffold and / or CDR residues within the Vh and / or Vl domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the scaffold and / or CDR residues are mutated to the corresponding residue (or residues) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the scaffold and / or CDR regions, for example, where certain individual residues are mutated to the... Petition 870260046565, dated 05 / 15 / 2026, p. 68 / 435 60 / 202 corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be easily tested for one or more desired properties such as enhanced binding specificity, increased binding (e.g., as measured by cell binding titration or FACS binding) or binding affinity (e.g., Kd), enhanced or intensified antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this manner are generally encompassed in the present invention.

[00139] The present invention also includes anti-MUC16 or anti-MUC16 / anti-CD3 antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the present invention includes anti-MUC16 or anti-MUC16 / anti-CD3 antibodies that have HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions with respect to any of the HCVR, LCVR, and / or CDR amino acid sequences shown in Table 1 herein or as described in Tables 16, 18, 19, 22, and 23 herein.

[00140] 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. A single antigen can have more than one epitope. Therefore, different Petition 870260046565, dated 05 / 15 / 2026, page 69 / 435 61 / 202 Antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is produced by adjacent amino acid residues in a polypeptide chain. Under certain circumstances, an epitope may include chemical portions of saccharides, phosphoryl groups, or sulfonyl groups in the antigen.

[00141] The term substantial identity or substantially identical, when referring to a nucleic acid or fragment thereof, indicates that, when ideally aligned with proper nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95% and, more preferably, at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by a well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule that has substantial identity with a reference nucleic acid molecule may, in certain cases, encode a polypeptide that has the same or substantially similar amino acid sequence to the polypeptide encoded by the reference nucleic acid molecule.

[00142] As applied to polypeptides, the term substantial similarity or substantially similar means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs using standard gap weighting, share at least 95% sequence identity, or even more preferably, at least 98% or 99% sequence identity. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions. A Petition 870260046565, dated 05 / 15 / 2026, p. 70 / 435 62 / 202 A conservative amino acid substitution is one in which one amino acid residue is replaced by another amino acid residue that has a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for performing this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Mol. Biol. methods 24: 307–331, incorporated herein by reference.Examples of amino acid groups 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) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninavalin, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the log PAM250 probability matrix revealed in Gonnet et al. (1992) Science 256: 1.443 a 1.445, incorporated herein by reference. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log probability matrix. Petition 870260046565, dated 05 / 15 / 2026, p. 71 / 435 63 / 202

[00143] Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures attributed 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 standard parameters to determine sequence homology or sequence identity between approximately related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutant of the same type. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with standard or recommended parameters, a program in GCG Version 6.1.FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using standard 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, each incorporated herein by reference. Germline Mutations

[00144] The anti-CD3 antibody disclosed herein comprises one or more amino acid substitutions, insertions and / or deletions in the scaffold and / or CDR regions of variable heavy chain domains compared to the lineage sequences. Petition 870260046565, dated 05 / 15 / 2026, p. 72 / 435 64 / 202 corresponding germline sequences from which the antibodies were derived.

[00145] The present invention also includes antibodies and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more scaffold and / or CDR regions are mutated to the corresponding residue (or residues) of the germline sequence from which the antibody was derived, or to the corresponding residue (or residues) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (or residues) (such sequence changes are referred to herein collectively as germline mutations), and which have weak or undetectable binding to a CD3 antigen.Several such exemplary antibodies that recognize CD3 are described in Tables 16, 18, 19, 22, and 23 in this document.

[00146] Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the scaffold and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are retained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be tested for one or more desired properties such as enhanced binding specificity, weak or reduced binding or binding affinity, enhanced or intensified pharmacokinetic properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments Petition 870260046565, dated 05 / 15 / 2026, page 73 / 435 65 / 202 antigens obtained in this manner, generally considering the orientation of the present disclosure, are encompassed within the present invention.

[00147] The present invention also includes anti-CD3 antibodies comprising variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-CD3 antibodies having HCVR, LCVR and / or CDR amino acid sequences, for example, with 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions with respect to any of the HCVR, LCVR and / or CDR amino acid sequences presented in Tables 16, 18, 19, 22 and 23 herein.The antibodies and bispecific antigen-binding molecules of the present invention comprise one or more amino acid substitutions, insertions, and / or deletions in the scaffold and / or CDR regions of the variable heavy and light chain domains compared to the corresponding germline sequences from which the individual antigen-binding domains were derived, while maintaining or enhancing the desired weak to undetectable binding to the CD3 antigen. A conservative amino acid substitution is one in which an amino acid residue is replaced by another amino acid residue that has a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein; that is, the amino acid substitution maintains or enhances the desirable weak to undetectable binding or binding affinity in the case of anti-CD3 binding molecules. Examples of amino acid groups that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; Petition 870260046565, dated 05 / 15 / 2026, p. 74 / 435 66 / 202 (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) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups 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 revealed in Gonnet et al. (1992) Science 256: 1.443 to 1.445. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log probability matrix.

[00148] The present invention also includes antigen-binding molecules comprising an antigen-binding domain with an HCVR and / or CDR amino acid sequence that is substantially identical to any of the HCVR and / or CDR amino acid sequences disclosed herein, while maintaining or enhancing the desired weak affinity for CD3 antigen. The term substantial or substantially identical identity, in reference to an amino acid sequence, means that two amino acid sequences, when ideally aligned, such as through GAP or BESTFIT programs using standard gap weighting, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, the residue positions that are not identical differ by conservative amino acid substitutions.In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percent sequence identity or degree of similarity may be... Petition 870260046565, dated 05 / 15 / 2026, p. 75 / 435 67 / 202 adjusted upwards to correct for the conservative nature of the substitution. Methods for carrying out this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) methods Mol. Biol. 24: 307–331.

[00149] Sequence similarity for polypeptides, which is also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures attributed 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 standard parameters to determine sequence homology or sequence identity between approximately related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutant of the same type. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with standard or recommended parameters, a program in GCG Version 6.1.FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using standard parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403 to 410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389 to 3402.

[00150] Once obtained, the antigen-binding domains containing one or more germline mutations were tested. Petition 870260046565, dated 05 / 15 / 2026, page 76 / 435 68 / 202 regarding reduced binding or binding affinity using one or more in vitro assays. Although antibodies recognizing a particular antigen are typically examined for their purpose by testing for high (i.e., strong) binding or binding affinity to the antigen, the antibodies of the present invention exhibit weak or undetectable binding. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this manner are also encompassed in the present invention and have been found to be advantageous as avidity-driven tumor therapies.

[00151] Unexpected benefits, for example, improved pharmacokinetic properties and low patient toxicity, can be achieved from the methods described in this document. ANTIBODY BINDING PROPERTIES

[00152] As used in this document, the term binding in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein, for example, to a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.

[00153] For example, binding affinity typically corresponds to a Kd value of about 10⁻⁷ M or less, such as about 10⁻⁸ M or less, or about 10⁻⁹ M or less when determined, for example, by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using the antigen as the ligand and the antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand). Cell-based binding strategies, such as fluorescence-activated cell sorting (FACS) binding assays, are also used in this way. Petition 870260046565, dated 05 / 15 / 2026, p. 77 / 435 69 / 202 routine and FACS data correlate well with other methods such as radioligand competition ligation and SPR (Benedict, CA, J Immunol methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol methods. 2005, 302(1-2):68-77).

[00154] Consequently, the antibody or antigen-binding protein of the invention binds to the predetermined antigen or (receptor) cell surface molecule that has an affinity corresponding to a KD value that is at least ten times lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the present invention, an antibody affinity corresponding to a KD value that is equal to or less than ten times lower than a non-specific antigen may be considered undetectable binding; however, such an antibody may be paired with a second antigen-binding arm to produce a bispecific antibody of the invention.

[00155] The term KD (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen. There is an inverse relationship between KD and binding affinity, therefore, the lower the KD value, the higher, i.e., stronger, the affinity. Therefore, the terms higher affinity or stronger affinity refer to a superior ability to form an interaction and therefore a lower KD value, and conversely, the terms lower affinity or weaker affinity refer to a lower ability to form an interaction and therefore a higher KD value.In some circumstances, a higher binding affinity (or KD) of a particular molecule (e.g., antibody) to its interacting partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., antibody) to another interacting partner molecule (e.g., antigen X) may be present. Petition 870260046565, dated 05 / 15 / 2026, p. 78 / 435 70 / 202 antigen Y) can be expressed as a binding ratio determined by dividing a larger Kd value (lower or weaker affinity) by a smaller Kd value (higher or stronger affinity), for example, expressed as a binding affinity 5 times or 10 times greater, as the case may be.

[00156] The term kd (s -1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. This value is also called the koff value.

[00157] The term ka (M-1 x s-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[00158] The term Ka (M-1 or 1 / M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing ka by kd.

[00159] The term EC50 or EC50 refers to the half-effective maximum concentration, which includes the concentration of an antibody that induces an intermediate response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximum effect is observed. In certain embodiments, the EC50 value is equal to the concentration of an antibody of the invention that provides half-maximum binding to cells expressing CD3 or tumor-associated antigen, as determined, for example, by a FACS binding assay. Therefore, reduced or weaker binding is observed with an increased EC50, or half-effective maximum concentration value.

[00160] In one mode, the reduced connection can be defined Petition 870260046565, dated 05 / 15 / 2026, p. 79 / 435 71 / 202 as an antibody with an increased EC50 concentration that enables binding to half the maximum amount of target cells.

[00161] In another embodiment, the EC50 value represents the concentration of an antibody of the invention that elicits the half-maximum depletion of target cells by T-cell cytotoxicity activity. Therefore, increased cytotoxicity activity (e.g., T-cell-mediated tumor cell killing) is observed with a reduced EC50 or half-maximum effective concentration value. Bispecific Antigen-Binding Molecules

[00162] The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a 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 monospecific anti-MUC16 antibodies or bispecific anti-MUC16 / anti-CD3 antibodies of the present invention may be linked to or co-expressed with another functional molecule, for example, another peptide or protein.For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity or an additional binding specificity.

[00163] The use of the expression anti-CD3 antibody or anti-MUC16 antibody in this document is intended to include both monospecific anti-CD3 and anti-MUC16 antibodies, as well as bispecific antibodies comprising a CD3-binding arm and a MUC16-binding arm. Therefore, the present invention includes Petition 870260046565, dated 05 / 15 / 2026, p. 80 / 435 72 / 202 bispecific antibodies in which one immunoglobulin arm binds to human CD3, and the other immunoglobulin arm is specific for human MUC16. The CD3-binding arm may comprise any of the amino acid sequences of HCVR / LCVR or CDR as presented in Tables 1, 16, 18, 19, 22 and 23 in this document.

[00164] In certain embodiments, the CD3-binding arm binds to human CD3 and induces human T cell activation. In certain embodiments, the CD3-binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3-binding arm binds weakly to human CD3 and induces the killing of cells expressing tumor-associated antigen in the context of a bispecific or multispecific antibody. In other embodiments, the CD3-binding arm binds to or is weakly associated with human or cynomolgus (monkey) CD3, however, the binding interaction is not detectable by in vitro assays known in the art. The MUC16-binding arm may comprise any of the amino acid sequences of HCVR / LCVR or CDR as presented in Table 1 in this document.

[00165] According to certain exemplary embodiments, the present invention includes bispecific antigen-binding molecules that specifically bind to CD3 and MUC16. Such molecules may be referred to herein, for example, as bispecific anti-CD3 / anti-MUC16 molecules, or anti-CD3xMUC16 or CD3xMUC16 or other similar terminology (e.g., antiMUC16 / anti-CD3). The invention provides bispecific antigen-binding molecules constructed with a first antigen-binding arm that binds to MUC16 and a second antigen-binding arm that binds to CD3. In some embodiments, the antiCD3 arm comprises a heavy chain derived from IGHV3-9*01, Petition 870260046565, dated 05 / 15 / 2026, p. 81 / 435 73 / 202 IGHJ6*02, IGHD5-12*01. In other embodiments, the bispecific antigen-binding molecule activates human PBMC cells and / or induces cytotoxic activity in cell lines expressing tumor antigen.

[00166] The term MUC16, as used in this document, refers to the human MUC16 protein unless specified as being from a non-human species (e.g., mouse MUC16, monkey MUC16, etc.). The human MUC16 protein has the amino acid sequence shown in SEQ ID NO:1899.

[00167] The aforementioned bispecific antigen-binding molecules that specifically bind to CD3 and MUC16 may comprise an anti-CD3 antigen-binding molecule that binds to CD3 with a weak binding affinity, such as exhibiting a KD greater than about 40 nM, as measured by an in vitro affinity binding assay. The aforementioned bispecific antigen-binding molecules may comprise an anti-CD3 antigen-binding molecule that binds to CD3 and exhibits an EC50 greater than about 100 nM, as measured by a FACS titration assay.The aforementioned bispecific antigen-binding molecules may comprise an anti-CD3 antigen-binding molecule that does not exhibit any measurable or observable binding to CD3, as measured by an in vitro affinity binding assay or a FACS titration assay, yet retains the ability to activate human PBMC cells and / or induce cytotoxic activity in cell lines expressing tumor antigen.

[00168] As used in this document, the term antigen-binding molecule means a protein, polypeptide or molecular complex comprising or consisting of at least one complementarity-determining region (CDR) that, alone or in combination with other molecules, is ... complementary protein-binding molecule. Petition 870260046565, dated 05 / 15 / 2026, page 82 / 435 74 / 202, in combination with one or more additional CDRs and / or scaffold regions (FRs), specifically binds to a particular antigen. In certain embodiments, an antigen-binding molecule is an antibody or a fragment of an antibody, as those terms are defined elsewhere in this document.

[00169] As used in this document, the expression bispecific antigen-binding molecule means a protein, polypeptide, or molecular complex comprising at least one first antigen-binding domain and a second antigen-binding domain. Each antigen-binding domain within the bispecific antigen-binding molecule comprises 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, the first antigen-binding domain specifically binds to a first antigen (e.g., CD3) and the second antigen-binding domain specifically binds to a distinct second antigen (e.g., MUC16).

[00170] 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 comprises 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 (e.g., a bispecific antibody), the CDRs of the first antigen-binding domain may be designated with the prefix A1 and the CDRs of the second antigen-binding domain may be designated with the prefix A2. Therefore, the CDRs of the first antigen-binding domain may be referred to in this document as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDRs of the second antigen-binding domain as A2. Petition 870260046565, dated 05 / 15 / 2026, page 83 / 435 75 / 202 antigens may be referred to in this document as A2-HCDR1, A2-HCDR2 and A2-HCDR3.

[00171] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form a bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerization domain. The association of one multimerization domain with another multimerization domain facilitates the association between the two antigen-binding domains, thus forming a bispecific antigen-binding molecule. As used herein, a multimerization domain is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of equal or similar structure or constitution. For example, a multimerization domain can be a polypeptide comprising an immunoglobulin CH3 domain.A non-limiting example of a multimerization component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), for example, an Fc domain of an IgG selected from the IgG1, IgG2, IgG3, and IgG4 isotypes, as well as any allotype within each isotype group.

[00172] The bispecific antigen-binding molecules of the present invention will typically comprise two multimerization domains, for example, two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerization domains Petition 870260046565, dated 05 / 15 / 2026, page 84 / 435 76 / 202 can be different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[00173] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[00174] Any bispecific antibody format or technology can be used to produce the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally 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 having a second antigen-binding specificity, to produce a bispecific antigen-binding molecule.Specific exemplary bispecific formats that may be used in the context of the present invention include, without limitation, for example, diabody or scFv-based bispecific formats, IgG-scFv fusions, dual variable domain (DVD) Ig, Quadroma, button-in-hole packaging, common light chain (e.g., common light chain with button-in-hole packaging, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab2 bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1 to 11, and references cited herein, for a review of the aforementioned formats). Petition 870260046565, dated 05 / 15 / 2026, page 85 / 435 77 / 202

[00175] In the context of bispecific antigen-binding molecules of the present invention, the multimerization domains, for example, Fc domains, may comprise one or more amino acid alterations (e.g., insertions, deletions, or substitutions) compared to the naturally occurring wild-type version of the Fc domain. For example, the invention includes bispecific antigen-binding molecules comprising one or more modifications to the Fc domain resulting in a modified Fc domain that has a modified (e.g., enhanced or reduced) binding interaction between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a modification in a Ch2 or Ch3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or a modification at position 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or a modification at position 250 and / or 428; or a modification at position 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modification comprises the 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a modification of 250Q and 428L (e.g., T250Q and M428L); and a modification of 307 and / or 308 (e.g., 308F or 308P).

[00176] The present invention also includes molecules for binding to Petition 870260046565, dated 05 / 15 / 2026, p. 86 / 435 78 / 202 bispecific antigen comprising a first Ch3 domain and a second Ig Ch3 domain, wherein the first and second Ig Ch3 domains differ from each other by at least one amino acid and wherein at least one amino acid difference reduces the binding of the bispecific antibody to Protein A compared with a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig Ch3 domain binds to Protein A and the second Ig Ch3 domain contains a mutation that reduces or abolishes Protein A binding as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second Ch3 may additionally comprise a Y96F modification (by IMGT; Y436F by EU). See, for example, U.S. Patent No. 8,586,713.Additional modifications that may be found within the second Ch3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies.

[00177] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise part or all of a Ch2 sequence derived from a Ch2 region of human IgG1, human IgG2, or human IgG4, and part or all of a Ch3 sequence derived from a human IgG1, human IgG2, or human IgG4. A chimeric Fc domain may also contain a chimeric linkage region.For example, a chimeric articulation may comprise a superior articulation sequence, derived from an articulation region of a human IgG1, a human IgG2, or a human IgG4, combined. Petition 870260046565, dated 05 / 15 / 2026, page 87 / 435 79 / 202 with a lower articulation sequence, derived from an articulation region of a human IgG1, a human IgG2, or a human IgG4. A particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules presented herein comprises, from the N-terminus to the C-terminus: [Ch1 of IgG4] - [upper articulation of IgG4] - [lower articulation of IgG2] - [CH2 of IgG4] - [CH3 of IgG4]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules presented herein comprises, from the N-terminus to the C-terminus: [Ch1 of IgG1] - [upper articulation of IgG1] - [lower articulation of IgG2] - [CH2 of IgG4] - [CH3 of IgG1]. These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in a US Publication.2014 / 0243504, published on August 28, 2014, which is incorporated into this document in its entirety. Chimeric Fc domains that have these general structural arrangements, and variants thereof, may have altered Fc receptor binding, which, in turn, affects Fc effector function.

[00178] In certain embodiments, the invention provides an antibody heavy chain wherein the heavy chain constant (CH) region comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NO: 1911, SEQ ID NO: 1912, SEQ ID NO: 1913, SEQ ID NO: 1914, SEQ ID NO: 1915, SEQ ID NO: 1916, SEQ ID NO: 1917, SEQ ID NO: 1918, SEQ ID NO: 1919, or SEQ ID NO: 1920. In some embodiments, the heavy chain constant (CH) region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1911, SEQ ID NO: 1912, SEQ ID NO: 1913, SEQ ID NO: 1914, SEQ ID NO: 1915, SEQ ID NO: Petition 870260046565, dated 05 / 15 / 2026, p. 88 / 435 80 / 202 1916, SEQ ID NO: 1917, SEQ ID NO: 1918, SEQ ID NO: 1919 and SEQ ID NO: 1920.

[00179] In other embodiments, the invention provides an antibody heavy chain wherein the Fc domain comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NO: 1921, SEQ ID NO: 1922, SEQ ID NO: 1923, SEQ ID NO: 1924, SEQ ID NO: 1925, SEQ ID NO: 1926, SEQ ID NO: 1927, SEQ ID NO: 1928, SEQ ID NO: 1929, or SEQ ID NO: 1930. In some embodiments, the Fc domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1921, SEQ ID NO: 1922, SEQ ID NO: 1923, SEQ ID NO: 1924, SEQ ID NO: 1925, SEQ ID NO: 1926, SEQ ID NO: 1927, SEQ ID NO: 1928, SEQ ID NO: 1929 and SEQ ID NO: 1930. SEQUENCE VARIANTS

[00180] The antibodies and bispecific antigen-binding molecules of the present invention may comprise one or more amino acid substitutions, insertions, and / or deletions in the scaffold and / or CDR regions of the variable heavy and light chain domains compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present invention may comprise antigen-binding domains that are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more regions of Petition 870260046565, dated 05 / 15 / 2026, p. 89 / 435 81 / 202 framework and / or CDR are mutated to the corresponding residue (or residues) of the germline sequence from which the antibody was derived, or to the corresponding residue (or residues) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (or residues) (such sequence alterations are referred to herein collectively as germline mutations). A person of ordinary skill in the art, beginning with the variable region sequences of heavy and light chains disclosed herein, can readily produce numerous antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof.In certain embodiments, all scaffold and / or CDR residues within the Vh and / or Vl domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the scaffold and / or CDR residues are mutated to the corresponding residue (or residues) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).Furthermore, antigen-binding domains can contain any combination of two or more germline mutations within the scaffold and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of. Petition 870260046565, dated 05 / 15 / 2026, page 90 / 435 82 / 202 a particular germline sequence while certain other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains containing one or more germline mutations can be easily tested for one or more desired properties such as enhanced binding specificity, increased binding or binding affinity, enhanced or intensified antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this manner are generally encompassed within the present invention.

[00181] The present invention also includes antigen-binding molecules in which one or both antigen-binding domains comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the present invention includes antigen-binding molecules comprising an antigen-binding domain having HCVR, LCVR, and / or CDR amino acid sequences, for example, with 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions with respect to any of 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 that has a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).In general, a conservative amino acid substitution will not substantially alter the functional properties. Petition 870260046565, dated 05 / 15 / 2026, p. 91 / 435 83 / 202 of a protein. Examples of amino acid groups 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) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups 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 log PAM250 probability matrix revealed in Gonnet et al. (1992) Science 256: 1.443 a 1.445, incorporated herein by reference. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log likelihood matrix.

[00182] The present invention also includes antigen-binding molecules comprising an antigen-binding domain with an HCVR, LCVR, and / or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. The term substantial or substantially identical identity, in reference to an amino acid sequence, means that two amino acid sequences, when ideally aligned, such as through the GAP or BESTFIT programs using standard gap weightings, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.Preferably, residue positions that are not identical differ by amino acid substitutions. Petition 870260046565, dated 05 / 15 / 2026, p. 92 / 435 84 / 202 conservative. In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means of performing this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Mol. Biol. methods 24: 307–331, incorporated herein by reference.

[00183] Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures attributed 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 standard parameters to determine sequence homology or sequence identity between approximately related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutant of the same type. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with standard or recommended parameters, a program in GCG Version 6.1.FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP or TBLASTN, using standard parameters. Query, for example, Petition 870260046565, dated 05 / 15 / 2026, page 93 / 435 85 / 202 Altschul et al. (1990) J. Mol. Biol. 215:403 to 410 and Altschul et al. (1997) Nucleic Acids Res. 25:3,389 to 3,402, each incorporated herein by reference. pH-dependent bonding

[00184] The present invention includes anti-MUC16 antibodies and bispecific anti-CD3 / anti-MUC16 antigen-binding molecules with pH-dependent binding characteristics. For example, an anti-MUC16 antibody of the present invention may exhibit reduced binding to MUC16 at acidic pH compared to neutral pH. Alternatively, the anti-MUC16 antibodies of the invention may exhibit enhanced binding to MUC16 at acidic pH compared to neutral pH. The term 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 in this document, the term neutral pH means a pH from about 7.0 to about 7.4. The term 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.

[00185] In certain cases, reduced binding at acidic pH compared to neutral pH is expressed in terms of a ratio of the KD value of antibody binding to its antigen at acidic pH to the KD value of antibody 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 MUC16 at acidic pH compared to neutral pH for the purposes of the present invention if the antibody or antigen-binding fragment thereof exhibits an acidic / neutral KD ratio of about 3.0 or greater. In certain exemplary embodiments, the acidic / 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, Petition 870260046565, dated 05 / 15 / 2026, page 94 / 435 86 / 202 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 higher.

[00186] Antibodies with pH-dependent binding characteristics can be obtained, for example, by examining a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH compared to neutral pH. Additionally, modifications of the antigen-binding domain at the amino acid level can yield antibodies with pH-dependent characteristics. For example, by replacing one or more amino acids in an antigen-binding domain (e.g., within a CDR) with a histidine residue, an antibody with reduced antigen binding at acidic pH compared to neutral pH can be obtained. Antibodies comprising variants of Fc

[00187] According to certain embodiments of the present invention, anti-MUC16 antibodies and bispecific anti-CD3 / anti-MUC16 antigen-binding molecules are provided comprising an Fc domain comprising one or more mutations that enhance or diminish antibody binding to the FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the present invention includes antibodies comprising a mutation in the CH2 or CH3 region of the Fc domain, wherein the mutation (or mutations) increases the affinity of the Fc domain to the FcRn in an acidic environment (for example, in an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in the serum half-life of the antibody when administered to an animal.Non-limiting examples of such Fc modifications include, for example, a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or a... Petition 870260046565, dated 05 / 15 / 2026, p. 95 / 435 87 / 202 modification to heading 428 and / or 433 (for example, H / L / R / S / P / Q or K) and / or 434 (for example, H / F or Y); or a modification to heading 250 and / or 428; or a modification to heading 307 or 308 (for example, 308F, V308F) and 434. In one embodiment, the modification comprises the 428L (for example, M428L) and 434S (for example, N434S) modification; a 428L, 259I (for example, V259I), and 308F (for example, V308F) modification; a 433K (for example, H433K) and a 434 (for example, 434Y) modification; a 252, 254 and 256 (e.g., 252Y, 254T and 256E) modification; a modification of 250Q and 428L (e.g., T250Q and M428L); and a modification of 307 and / or 308 (e.g., 308F or 308P).

[00188] For example, the present invention includes anti-MUC16 antibodies and bispecific anti-CD3 / anti-MUC16 antigen-binding molecules comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present invention. BIOLOGICAL CHARACTERISTICS OF ANTIBODIES AND BINDING MOLECULES TO THE BIOSPECIFIC ANTIGEN

[00189] The present invention includes antibodies and antigen-binding fragments thereof that bind to human MUC16 with high affinity (e.g., subnanomolar KD values).

[00190] According to certain embodiments, the present invention includes antibodies and antibody antigen-binding fragments that bind to human MUC16 (e.g., at 25 °C) with a lower Kd. Petition 870260046565, dated 05 / 15 / 2026, p. 96 / 435 88 / 202 than about 60 nM as measured by surface plasmon resonance, for example, using an assay format as defined in Example 4 in this document.In certain embodiments, the antibodies or antigen-binding fragments of the present invention bind to MUC16 with a Kd less than about 60 nM, less than about 40 nM, less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, less than about 500 pM, less than about 400 pM, or less than about 300 pM, as measured by surface plasmon resonance, for example, using an assay format as defined in Example 4 herein (e.g., mAb capture format). or antigen capture) or a substantially similar assay.The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies that bind to MUC16 with a KD less than about 7 nM, as measured by surface plasmon resonance, for example, using an assay format as defined in Example 4 herein (e.g., mAb capture or antigen capture format), or a substantially similar assay.

[00191] The present invention also includes antibodies and antigen-binding fragments thereof that bind to MUC16 with a dissociative half-life (0½) greater than about 10 minutes or greater than about 125 minutes, as measured by surface plasmon resonance at 25 °C, for example, using an assay format as defined in Example 4 herein, or a substantially similar assay. In certain embodiments, antibodies or Petition 870260046565, dated 05 / 15 / 2026, p. 97 / 435 89 / 202 antigen-binding fragments of the present invention bind to MUC16 with a ½ greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 110 minutes, or greater than about 120 minutes, as measured by surface plasmon resonance at 25 °C, for example, using an assay format as defined in Example 4 herein (e.g., mAb capture or antigen capture format), or a substantially similar assay.The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies that bind to MUC16 with a 1½ greater than about 10 minutes or greater than about 20 minutes as measured by surface plasmon resonance at 25 °C, for example, using an assay format as defined in Example 4 herein, or a substantially similar assay.

[00192] The present invention also includes antibodies and antigen-binding fragments thereof that bind specifically to human cell lines expressing endogenous MUC16 (e.g., OVCAR-3), as determined by an electrochemiluminescence-based detection assay as presented in Example 2 or a substantially similar assay.

[00193] The present invention also includes bispecific antigen-binding molecules anti-CD3 / anti-MUC16 that exhibit one or more features selected from the group consisting of: (a) inhibiting tumor growth in immunocompromised mice bearing human ovarian cancer xenografts; and (b) suppressing tumor growth of established tumors in mice. Petition 870260046565, dated 05 / 15 / 2026, p. 98 / 435 90 / 202 immunocompromised individuals carrying human ovarian cancer xenografts (see, for example, Example 8).

[00194] The present invention includes antibodies and antigen-binding fragments thereof that bind to human CD3 with high affinity. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD3 with medium or low affinity, depending on the therapeutic context and particular targeting properties that are desired. In some cases, low affinity includes antibodies that bind to CD3 with a Kd or EC50 (e.g., as measured in a surface plasmon resonance assay) greater than 300 nM, greater than 500 nM, or greater than 1 μM. The present invention also includes antibodies and antigen-binding fragments thereof that bind to human CD3 without measurable affinity.For example, in the context of a bispecific antigen-binding molecule, where one arm binds to CD3 and the other arm binds to a target antigen (e.g., MUC16), it may be desirable for the target antigen-binding arm to bind to the target antigen with high affinity while the anti-CD3 arm binds to CD3 only with moderate or low affinity or no affinity at all. In this way, preferential targeting of the antigen-binding molecule to cells expressing the target antigen can be achieved while avoiding binding to general / non-targeted CD3 and the consequent adverse side effects associated with it.

[00195] The present invention includes bispecific antigen-binding molecules (e.g., bispecific antibodies) that are capable of simultaneously binding to human CD3 and human MUC16. The binding arm that interacts with cells expressing CD3 may have weak to undetectable binding as measured in a suitable in vitro binding assay. The extent to which a molecule Petition 870260046565, dated 05 / 15 / 2026, p. 99 / 435 The 91 / 202 bispecific antigen binding to cells expressing CD3 and / or MUC16 can be assessed by fluorescence activated cell sorting (FACS), as illustrated in Example 5 in this document.

[00196] For example, the present invention includes antibodies, antigen-binding fragments and bispecific antibodies thereof that bind specifically to human T cell lines expressing CD3 but not expressing MUC16 (e.g., Jurkat), primate T cells (e.g., peripheral blood cynomolgus mononuclear cells [PBMCs]) and / or cells expressing MUC16.

[00197] The present invention includes antibodies, antigen-binding fragments and bispecific antibodies thereof that bind to human CD3 with weak (i.e., low) binding or binding affinity or even undetectable binding or binding affinity.

[00198] The present invention includes antibodies, antigen-binding fragments and bispecific antibodies thereof that bind to monkey CD3 (i.e., cynomolgus) CD3 with weak (i.e., low) binding or binding affinity or even undetectable binding or binding affinity.

[00199] The present invention includes antibodies, antigen-binding fragments and bispecific antibodies thereof that bind to human CD3 and induce T cell activation.

[00200] The present invention includes bispecific anti-CD3 / anti-MUC16 antigen-binding molecules that are capable of depleting antigen-expressing tumor cells in an individual (see, for example, Example 8, in a bioluminescent imaging assay or a substantially similar assay). For example, according to certain embodiments, bispecific anti-CD3 / anti-MUC16 antigen-binding molecules are provided, in which a single administration Petition 870260046565, dated 05 / 15 / 2026, p. 100 / 435 92 / 202 of 10 pg of the bispecific antigen-binding molecule to an individual causes a reduction in the number of cells expressing MUC16 in the individual (e.g., tumor growth in the individual is suppressed or inhibited). Unless otherwise indicated, bioluminescent radiance refers to [p / s / cm22 / sr].

[00201] The present invention also includes anti-MUC16 antibody-drug conjugates that inhibit tumor growth in MUC16-positive ovarian cancer xenograft models in vivo (see, for example, Example 10, in a bioluminescent imaging assay or a substantially similar assay). In certain embodiments, the anti-MUC16 antibody-drug conjugates with Compound 7 are provided, wherein four once-weekly doses administered at a dose of 85 pg / kg inhibit intraperitoneal OVCAR3 / luc tumor growth in vivo. In certain embodiments, the anti-MUC16 antibody-drug conjugates with Compound 7 are provided, wherein four once-weekly doses administered at a dose of 85 pg / kg inhibit subcutaneous OVCAR3 / luc tumor growth in vivo.In certain embodiments, anti-MUC16 antibody-drug conjugates with Compound 10 are provided, wherein a single dose at a dose of 85 pg / kg, 170 pg / kg or 340 pg / kg inhibits intraperitoneal OVCAR3 / luc tumor growth in vivo. Unless otherwise indicated, bioluminescent radiance refers to [p / s / cm22 / sr].

[00202] The present invention also includes bispecific antigen-binding molecules anti-CD3 / anti-MUC16 that exhibit pharmacokinetic profiles in humanized MUC16 x CD3 mice (mice homozygous for human MUC16 and CD3 expression, MUC16hu / hu x CD3hu / hu), humanized CD3 mice (mice homozygous for human CD3 expression, CD3hu / hu), and wild-type (WT) mice of the corresponding strain. Petition 870260046565, dated 05 / 15 / 2026, p. 101 / 435 93 / 202 (75% C57BL, 25% 129Sv), as described in Example 7 and shown in Figures 1, 2 and 3. EPITOPE MAPPING AND RELATED TECHNOLOGIES

[00203] The epitope on CD3 and / or MUC16 to which the antigen-binding molecules of the present invention bind may consist of a single 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 from a CD3 or MUC16 protein. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) from CD3 or MUC16. The antibodies of the invention can interact with amino acids contained within a single CD3 chain (e.g., CD3-epsilon, CD3-delta, or CD3-gamma), or they can interact with amino acids in two or more different CD3 chains. The term epitope, as used herein, 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. A single antigen may have more than one epitope.Therefore, different antibodies can bind to different areas on an antigen and may have different biological effects. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Under certain circumstances, an epitope may include chemical portions of saccharides, phosphoryl groups, or sulfonyl groups in the antigen.

[00204] Several techniques known to people of ordinary technical skill can be used to determine whether an antigen-binding domain of an antibody interacts with one or more amino acids within a polypeptide or protein. The techniques Petition 870260046565, dated 05 / 15 / 2026, page 102 / 435 Examples include, for instance, routine cross-block assays such as that described in Antibodies, Harlow, and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine-scan mutational analysis, peptide labeling analysis (Reineke, 2004, Mol Biol methods 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which an antibody's antigen-binding domain interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium identification of the protein of interest, followed by antibody binding to the deuterium-identified protein.Next, the protein / antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for those protected by the antibody (which remain deuterium-tagged). After antibody dissociation, the target protein is subjected to protease cleavage and mass spectrometry analysis, thus revealing the deuterium-tagged 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 crystallography of the antigen / antibody complex can also be used for epitope mapping purposes.

[00205] The present invention further includes antiMUC16 antibodies that bind to the same epitope as any of the specific exemplary antibodies described herein (for example, antibodies comprising any of the amino acid sequences as presented in Table 1). Petition 870260046565, dated 05 / 15 / 2026, p. 103 / 435 95 / 202 in this document). Similarly, the present invention also includes anti-MUC16 antibodies that compete for binding to MUC16 with any of the specific exemplary antibodies described in this document (for example, antibodies comprising any of the amino acid sequences as presented in Table 1 in this document).

[00206] The present invention also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that binds specifically to human CD3 and / or cynomolgus CD3 with low or undetectable binding or binding affinity, and a second antigen-binding domain that binds specifically to human MUC16, wherein the first antigen-binding domain binds to the same epitope on CD3 as any of the specific exemplary CD3-specific antigen-binding domains described herein and / or wherein the second antigen-binding domain binds to the same epitope on MUC16 as any of the specific exemplary MUC16-specific antigen-binding domains described herein.

[00207] Similarly, the present invention also includes bispecific antigen-binding molecules comprising a first antigen-binding domain that specifically binds to human CD3, and a second antigen-binding domain that specifically binds to human MUC16, wherein the first antigen-binding domain competes for binding to CD3 with any of the specific exemplary CD3-specific antigen-binding domains described herein, and / or wherein the second antigen-binding domain competes for binding to MUC16 with any of the specific exemplary MUC16-specific antigen-binding domains described herein. Petition 870260046565, dated 05 / 15 / 2026, p. 104 / 435 96 / 202 document.

[00208] It is readily possible to determine whether a particular antigen-binding molecule (e.g., antibody) or antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule of the present invention using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope on MUC16 (or CD3) as a reference bispecific antigen-binding molecule of the present invention, the bispecific reference molecule may first bind to a MUC16 protein (or CD3 protein). Then, the ability of a test antibody to bind to the MUC16 (or CD3) molecule is evaluated.If the test antibody can bind to MUC16 (or CD3) after saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different MUC16 (or CD3) epitope than the reference bispecific antigen-binding molecule. Conversely, if the test antibody cannot bind to the MUC16 (or CD3) molecule after saturation binding with the reference bispecific antigen-binding molecule, then the test antibody may bind to the same MUC16 (or CD3) epitope as the epitope bound by the reference bispecific antigen-binding molecule of the invention.Further routine experimentation (e.g., mutation and peptide binding analyses) can then be performed to confirm whether the observed lack of binding of the test antibody is, in fact, due to binding to the same epitope as the reference bispecific antigen-binding molecule or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. Experiments of this type can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative antibody binding assay. Petition 870260046565, dated 05 / 15 / 2026, page 105 / 435 97 / 202 or qualitative information available in the art. According to certain embodiments of the present invention, two antigen-binding proteins bind to the same (or overlapping) epitope if, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antigen-binding protein inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99% as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res.). Alternatively, two antigen-binding proteins are considered to bind to the same epitope if essentially 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 if only a subset of the amino acid mutations that reduce or eliminate binding in one antigen-binding protein also reduce or eliminate binding in the other.

[00209] To determine whether an antibody or its antigen-binding domain competes for binding with a reference antigen-binding molecule, the binding methodology described above is performed in two orientations: In a first orientation, the reference antigen-binding molecule is allowed to bind to a MUC16 protein (or CD3 protein) under saturation conditions, followed by evaluation of the binding of the test antibody to the MUC16 (or CD3) molecule. In a second orientation, the test antibody is allowed to bind to a MUC16 (or CD3) molecule under saturation conditions, followed by evaluation of the binding of the reference antigen-binding molecule to the MUC16 (or CD3) molecule. If, in both orientations, only the first (saturation) antigen-binding molecule is able to bind to the MUC16 (or CD3) molecule, then it is concluded that the test antibody and the antigen-binding molecule are competing for binding. Petition 870260046565, dated 05 / 15 / 2026, p. 106 / 435 Reference 98 / 202 antibodies compete for binding to MUC16 (or CD3). As will be observed by a person of ordinary skill in the art, an antibody that competes for binding with a reference antigen-binding molecule does not necessarily bind to the same epitope as the reference antibody; however, it can sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope. Preparation of antigen-binding domains and construction of bispecific molecules.

[00210] Antigen-binding domains specific for particular antigens can be prepared by any antibody-generating technology known in the art. Once obtained, two different antigen-binding domains, specific for two different antigens (e.g., CD3 and MUC16), can be suitably arranged relative to each other to produce a bispecific antigen-binding molecule of the present invention using routine 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, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules of the invention are derived from chimeric, humanized, or fully human antibodies.The methods for producing 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 prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody-generating technology), high-affinity chimeric antibodies can be obtained. Petition 870260046565, dated 05 / 15 / 2026, page 107 / 435 Antibodies 99 / 202 to a particular antigen (e.g., CD3 or MUC16) are initially isolated, having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced by a desired human constant region to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules of the present invention.

[00211] Genetically modified animals can be used to produce bispecific human antigen-binding molecules. For example, a genetically modified mouse can be used which is unable to reassign and express an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operationally linked to the mouse kappa-type constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to produce fully human bispecific antigen-binding molecules comprising two different heavy chains that associate with an identical light chain comprising a variable domain derived from one of two different human light chain variable region gene segments. (See, for example, US patent 2011 / 0195454)."Wholly human" refers to an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by DNA derived from a human sequence along the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain thereof. In some cases, the sequence is entirely human. Petition 870260046565, dated 05 / 15 / 2026, page 108 / 435 100 / 202 human is derived from a protein endogenous to a human. In other cases, the fully human protein or protein sequence comprises a chimeric sequence in which each component sequence is derived from the human sequence. While not adhering to any theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenetic epitopes at component sequence junctions, for example, compared to any wild-type human immunoglobulin regions or domains. BIOEQUIVALENTS

[00212] The present invention encompasses antigen-binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein, but retaining the ability to bind to CD3 and / or MUC16. Such variant molecules may comprise one or more amino acid additions, deletions, or substitutions compared to the parental sequence, but exhibit biological activity that is essentially equivalent to that of the bispecific antigen-binding molecules described.

[00213] The present invention includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules presented herein. Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, whether in single or multiple doses. Some antigen-binding proteins will be considered pharmaceutical equivalents or alternatives if they are equivalent in the extent of their absorption, but not in their rate of absorption. Petition 870260046565, dated 05 / 15 / 2026, page 109 / 435 101 / 202 absorption and, however, can be considered bioequivalent since such differences in absorption rate are intentional and are reflected in the identification, are not essential for obtaining effective body drug concentrations, for example, in chronic use, and are considered medically insignificant for the particular drug product studied.

[00214] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

[00215] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can switch one or more times between the reference product and the biologic product without an expected increase in the risk of adverse effects, including a clinically significant alteration in immunogenicity or reduced efficiency, compared with continued therapy without such switching.

[00216] In one embodiment, two antigen-binding proteins are bioequivalent if both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.

[00217] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, for example, (a) an in vivo test in humans or other mammals in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the adequate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a controlled clinical trial that establishes the safety, efficacy, or bioavailability or bioequivalence of a Petition 870260046565, dated 05 / 15 / 2026, page 110 / 435 102 / 202 antigen-binding protein.

[00218] The bioequivalent variants of the exemplary bispecific antigen-binding molecules presented herein can be constructed, for example, by producing various substitutions of residues or sequences or by deleting terminal or internal residues or sequences not necessary for biological activity. For example, cysteine ​​residues not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges through renaturation. In other contexts, bioequivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules presented herein that comprise amino acid changes that modify the glycosylation characteristics of the molecules, for example, mutations that eliminate or remove glycosylation. Species selectivity and species cross-reactivity

[00219] According to certain embodiments of the invention, antigen-binding molecules are provided that bind to human CD3 but not to CD3 of other species. In addition, antigen-binding molecules are provided that bind to human MUC16. The present invention also includes antigen-binding molecules that bind to human CD3 and to CD3 of one or more non-human species; and / or antigen-binding molecules that bind to human MUC16.

[00220] According to certain exemplary embodiments of the invention, antigen-binding molecules are provided which bind to human CD3 and / or human MUC16 and may or may not bind, as the case may be, to one or more of CD3 and / or MUC16. Petition 870260046565, dated 05 / 15 / 2026, p. 111 / 435 103 / 202 of mouse, rat, guinea pig, cricket, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus monkey or chimpanzee. For example, in a particular exemplary embodiment, the bispecific antigen-binding molecules of the present invention are provided comprising a first antigen-binding domain that binds to human CD3 and cynomolgus CD3, and a second antigen-binding domain that binds specifically to human MUC16. ANTIBODY-DRUG CONJUGATES (ADCs)

[00221] The present invention provides antibody-drug conjugates (ADCs) comprising an antibody or antigen-binding fragment of the anti-MUC16 conjugated to a therapeutic chemical moiety such as a cytotoxic agent, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. In general terms, the ADCs comprise: A - [L - P]y, wherein A is an antigen-binding molecule, for example, an anti-MUC16 antibody or a fragment thereof (for example, a fragment comprising at least one HCDR3 selected from any of the HCDR3 amino acid sequences listed in Table 1), L is a ligand, P is the carrier or therapeutic chemical moiety (for example, cytotoxic agent), and y is an integer from 1 to 30.In several embodiments, the ADC comprises an anti-MUC16 antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and an LCVR having the amino acid sequences of the SEQ ID NOs (e.g., SEQ ID NOs: 2 and 10) shown in Table 1, or a specific HCVR / LCVR pair (e.g., SEQ ID NOs: 2 / 10). In some cases, the anti-MUC16 antibody or fragment comprises CDRs with the amino acid sequences of the SEQ ID NOs (e.g., SEQ ID NOs: 4-6-8-12-14-16) shown in Table 1. In some cases, the anti-MUC16 antibody or fragment comprises an HCVR and a... Petition 870260046565, dated 05 / 15 / 2026, p. 112 / 435 104 / 202 LCVR that has the amino acid sequences of the SEQ ID NOs (e.g., SEQ ID NOs: 2 and 10) shown in Table 1, or a pair of specific amino acid sequences (e.g., SEQ ID NOs: 2 / 10). In some cases, the anti-MUC16 antibody is an antibody or antigen-binding fragment that binds to human MUC16 within one or more of five proximal membrane SEA domains of human MUC16 corresponding to residues 13,791 to 14,451 of SEQ ID NO: 1899. In some cases, the anti-MUC16 antibody is an antibody or antigen-binding fragment that binds to human MUC16 within residues 13,810 to 14,451 of SEQ ID NO: 1899. In some cases, the anti-MUC16 antibody is an antibody or antigen-binding fragment that binds to any one or more of SEA1, SEA2, SEA3, SEA4, SEA5, SEA6, SEA7, SEA8, SEA9, SEA10, SEA11, SEA12, SEA13, SEA14, SEA15, or SEA16 of Human MUC16.

[00222] Cytotoxic agents include any agent that is detrimental to the growth, viability, or propagation of cells. The antigen-binding molecules or antibodies of the invention deliver these cytotoxic agents, referred to herein as payloads, to target cells. Examples of suitable cytotoxic agents and therapeutic chemoagents for forming ADCs are known in the art.

[00223] Examples of suitable cytotoxic agents and therapeutic chemoagents that can be conjugated to anti-MUC16 antibodies according to this aspect of the invention include, for example, 1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-di-yne-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitins, aminopterin, anguidin, anthracycline, anramycin (AMC), auristatins (monomethyl auristatin E or monomethyl auristatin F), bleomycin, busulfan, acid Petition 870260046565, dated 05 / 15 / 2026, page 113 / 435 105 / 202 butyric acid, calicheamycins, camptothecin, carminomycins, carmustine, cemadotins, cisplatin, colchicine, combretastatins, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine, diacetoxypentyldoxorubicin, dibromomannitol, dihydroxyanthracinedione, disorazois, dolastatin, doxorubicin, duocarmycin, echinomycins, heuterines, emetine, epothilones, spermycin, estramine, ustines, ethidium bromete, etoposide, fluorouracils, geldanamycins, gramicidin D, glucocorticoids, irinotecans, leptomycins, leurosines, lidocaine, lomustine (CCNU), maitansinoids, mechlorethamine, melphalan mercaptopurines, methoturins, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetylspermidine, podophyllotoxins, procaine, propranolol, pteridines, puromycin, rhizoxins, streptozotocin, talismomycins, taxol, tenoposide, tetracaine, thioepa, chlorambucil, tomaimides, topothecans, tubulin, vinblastine, vincristine, vindesinevinorelbines and derivatives of any of the aforementioned substances.

[00224] According to certain embodiments, the cytotoxic agent conjugated to an anti-MUC16 antibody is an auristatin such as monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), a tubulisin such as TUB-OH or TUB-OMOM, a tomaimycin derivative, a dolastatin derivative, or a maytansinoid such as DM1 or DM4. In some embodiments, the cytotoxic agent is a maytansinoid having the structure of Formula (I), including the stereoisomers of the compounds of Formula (I): (FORMULA I) Petition 870260046565, dated 05 / 15 / 2026, p. 114 / 435 106 / 202 where A is arylene or heteroarylene.

[00225] In some embodiments, A is a divalent radical of benzene, pyridine, naphthalene or quinolone, which are optionally substituted.

[00226] In some forms, A is arylene.

[00227] In some modalities, A is:

[00228] where:

[00229] R1 is, independently, in each occurrence, alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, ^'orA, ψ5θ2κA, -^-r* or azido,

[00230] where RA is alkyl or heteroalkyl;

[00231] n is an integer from 0 to 4;

[00232] m is an integer from 0 to 3;

[00233] p is an integer from 0 to 6; and

[00234] q is an integer from 0 to 5.

[00235] In some forms, the Formula 1 compound is selected from the group consisting of: Petition 870260046565, dated 05 / 15 / 2026, p. 115 / 435 107 / 202 Petition 870260046565, dated 05 / 15 / 2026, p. 116 / 435 108 / 202 Petition 870260046565, dated 05 / 15 / 2026, p. 117 / 435 109 / 202

[00237] In some embodiments, the maytansinoid of Formula (I) is conjugated to an anti-MUC16 antibody or antigen-binding fragment thereof by means of a linker, as shown in Formula (IA), below: Petition 870260046565, dated 05 / 15 / 2026, p. 118 / 435 110 / 202 (FORMULA IA)

[00238] where:

[00239] A is arylene or heteroarylene, as discussed above in relation to Formula (I);

[00240] L is a ligand;

[00241] BA is an anti-MUC16 antibody or antigen-binding fragment thereof; and

[00242] k is an integer from 1 to 30.

[00243] In several modalities, L is: THE 4-SP-AA1-AA2-^

[00244] where:

[00245] SP is a spacer;

[00246] is one or more binding sites to anti-MUC16 antibody or a fragment thereof;

[00247] AA1 is an amino acid; and

[00248] AA2 is an amino acid.

[00249] In some embodiments, AA1-AA2 is: valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine, asparagine, asparagine-valine, threonine-asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid-asparagine, glutamic acid of asparagine, Petition 870260046565, dated 05 / 15 / 2026, page 119 / 435 111 / 202 citrulline-asparagine, asparagine-citrulline, alanine-asparagine or asparagine-alanine.

[00250] In some sports, SP is: ο ο N-(CH2)b-Mο or

[00251] where:

[00252] « is a link to anti-MUC16 antibody or a fragment thereof; and

[00253] b is an integer from 2 to 8.

[00254] In other modalities, L is:

[00255] where:

[00256] « is a link to anti-MUC16 antibody or a fragment thereof; and

[00257] b is an integer from 2 to 8.

[00258] In one embodiment, the compound of Formula (IA), including the ligand, which is linked to the anti-MUC16 antibody or antigen-binding fragment thereof is: Petition 870260046565, dated 05 / 15 / 2026, p. 120 / 435 112 / 202 where « is a link to the anti-MUC16 antibody or a fragment thereof. In some cases, this chemical portion is called Compound 10.

[00259] In one embodiment, the compound of Formula (IA), including the ligand, which is linked to the anti-MUC16 antibody or antigen-binding fragment thereof is: where « is a link to the anti-MUC16 antibody or a fragment thereof. In some cases, this chemical portion is called Compound 60.

[00260] In some embodiments, the cytotoxic agent is a maytansinoid having the structure of Formula (II), including the stereoisomers of the compounds of Formula (II): Petition 870260046565, dated 05 / 15 / 2026, p. 121 / 435 113 / 202 (FORMULA II)

[00261] where:

[00262] A3a is an amino acid, a peptide having 2 to 20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -OC(=O)-O-, -OC(=O)-O-, -C(=O)-(CHx)p1-,-C(=O)O-(CHx)p1-, -(CHx)p1-C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-,((CH2)p2-O-)p3-, -C(=S)-,-C(=S)-S-, -C(=S)-NH-, -SC(=S)-, -SC(=S)-S-, -S-, -SO-, -SO2-, -NR4-,-N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)C(=O)-, -C(=O)-N(R4)-, C(=O)-N(R4)-C(=O)-, or -OC(=O)-NR4-, where alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are optionally substituted; and

[00263] p1, p2 and p3 are each independently 0, or an integer from 1 to 100;

[00264] x is 0, 1 or 2;

[00265] R4, R5, R6 and R8 are each independently H or a: alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl, substituted or unsubstituted; and

[00266] R4a is a: substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl group.

[00267] In some forms, the compound of Formula (II) is selected from the group consisting of: Petition 870260046565, dated 05 / 15 / 2026, p. 122 / 435 114 / 202

[00268] In one embodiment, the compound of Formula (II) is:

[00269] In some embodiments, the maytansinoid of Formula (II) is conjugated to an anti-MUC16 antibody or antigen-binding fragment thereof by means of a linker, as shown in Formula (IIA), below: Petition 870260046565, dated 05 / 15 / 2026, page 123 / 435 115 / 202

[00270]

[00271] BA-V- Z2—A—WX-AK (FORMULA IIA) where: BA is an anti-MUC16 antibody or antigen-binding fragment thereof;

[00272]

[00273] a is an integer from 1 to 30; Z2 is represented by the following structural formula: -Z2A-Z2BZ2C-Z2D, where Z2A, Z2B, Z2C and Z2D are, each independently, an amino acid, a peptide having from 2 to 20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -OC(=O)-, C(=O)-O-, -OC(=O)-O-, -C(=O)-(CHx)p1, -C(=O)-O-(CHx)p1, -(CHx)p1C(=O)-, -(CHx)p1-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, -C(=S)-, C(=S)-S-, -C(=S)-NH-, -SC(=S)-, -SC(=S)-S-, -S-, -SO-, -SO2-, -NR4-, N(R4)-C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, C(=O)-N(R4)-C(=O)-, -OC(=O)-N(R4), -OC(=S)-N(R4)-, -C(=S)-N(R4)-, o nA o n^\ -N=C=S, -N=C=O, o· or O ;

[00274] A is a natural or non-natural amino acid, or a peptide comprising from 2 to 20 amino acids;

[00275]

[00276] W is -O-, -S-, -CR5R6-, or -NR4-; X is aryl, heteroaryl, cycloalkyl, or heterocyclyl, wherein aryl, heteroaryl, cycloalkyl, and heterocyclyl are optionally Petition 870260046565, dated 05 / 15 / 2026, pp. 124 / 435 116 / 202 replaced;

[00277] where Ai, A3 and Ri are each independently an amino acid, a peptide having from 2 to 20 amino acids, an alkyl, an alkynyl, an alkenyl, a cycloalkyl, an aryl, a heteroaryl, a heterocyclyl, -CR5R6-, -O-, -C(=O)-, -OC(=O)-O-, -OC(=O)-O-, -C(=O)-(CHx)pi-, -C(=O)-O-(CHx)pi-, -(CHx)pi-C(=O)-, (CHx)pi-C(=O)-O-, -(O-(CH2)p2-)p3-, -((CH2)p2-O-)p3-, -C(=S)-, -C(=S)-S-, SC(=S)-, -C(=S)-NH-, -SC(=S)-S-, -S-, -SO-, -SO2-, -NR4-, -N(R4)C(=O)-N(R8)-, -N(R4)-C(=O)O-, -N(R4)-C(=O)-, -C(=O)-N(R4)-, -C(=O)N(R4)-C(=O)-, or -OC(=O)-NR4-, where alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl and heterocyclyl are optionally substituted;

[00278] R17 is selected from the group consisting of O, S, NRi8 and CR5R6;

[00279] Ri8 is selected from the group consisting of H, alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and acyl, in which alkyl, alkynyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl and acyl are optionally substituted;

[00280] R4, R5, R6 and R8 are, each independently, H or a: substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl; [0028i] R4a is a: substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl or heterocyclyl;

[00282] pi, p2 and p3 are each independently 0, or an integer from 1 to 100; and

[00283] x is 0, 1 or 2.

[00284] In some embodiments of Formula (IIA), A is a peptide selected from the group consisting of valine-citrulline, citrullinavaline, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine-asparagine, asparagine-threonine, serine Petition 870260046565, dated 05 / 15 / 2026, page 125 / 435 117 / 202 asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine and asparagine-alanine.

[00285] In one embodiment, the compound of Formula (IIA) that is linked to the anti-MUC16 antibody or antigen-binding fragment thereof is: -fem which is a link to the anti-MUC16 antibody or a fragment thereof. In some cases, this chemical portion is called Compound 7.

[00286] In some embodiments, the cytotoxic agent that is conjugated to an anti-MUC16 antibody or fragment thereof is a pure or substantially pure diastereomer of DM1: (DM1) ey is an integer from 1 to 0.

[00287] In another modality, the ADC comprises a structure of Petition 870260046565, dated 05 / 15 / 2026, p. 126 / 435 118 / 202 A - [L - P]y, where A is an anti-MUC16 antibody or antigen-binding fragment thereof, and [L - P] is:

[00288] a mixture of them, and

[00289] where y is an integer from 1 to 30, and

[00290] is a binding site to anti-MUC16 antibody or a fragment thereof.

[00291] Other maitansinoid derivatives are discussed in documents WO 2014 / 145090, WO2016 / 160615 and WO 2015 / 031396, each of which is incorporated herein by reference in its entirety.

[00292] In some embodiments, the cytotoxic agent that is conjugated to an anti-MUC16 antibody or fragment thereof is MMAE or MMAF.

[00293] Other cytotoxic agents known in the art are contemplated within the scope of the present invention, including, for example, protein toxins such as ricin, C. difficile toxin, Pseudomonas exotoxins, diphtheria toxin, botulinum toxin, briodin, saporin, pokeweed toxins (i.e., phytotoxin and phytolacigenin) Petition 870260046565, dated 05 / 15 / 2026, page 127 / 435 119 / 202 and others such as those presented in Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452 to 469.

[00294] Cytotoxic agents (cargoes) can be attached to an anti-MUC16 antigen-binding molecule or antibody of the invention by means of a chemical linker that covalently links the cargo compound to the protein molecule (i.e., antibody). Exemplary embodiments of specific linkers are discussed above. More generally, and as used herein, the term linker refers to any divalent chemical group or moiety that links, connects, or binds a binding agent (e.g., an antibody or an antigen-binding fragment thereof) with a cargo compound presented herein. Generally, suitable binding agent linkers for the antibody conjugates described herein are those that are sufficiently stable to exploit the circulating half-life of the antibody and, at the same time, capable of releasing their cargo after antigen-mediated internalization of the conjugate.Ligands can be cleavable or non-cleavable. Cleavable ligands are ligands that are cleaved by intracellular metabolism after internalization, for example, cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavable ligands are ligands that release an attached cargo via lysosomal degradation of the antibody after internalization. Suitable ligands include, but are not limited to, acid-labile ligands, hydrolysis-labile ligands, enzymatically cleavable ligands, reduction-labile ligands, autoimmolating ligands, and non-cleavable ligands. Suitable ligands also include, but are not limited to, those that are or comprise peptides, glucuronides, succinamide thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para units. Petition 870260046565, dated 05 / 15 / 2026, page 128 / 435 120 / 202 aminobenzyl (PAB). In some cases, the ligand is able to bind to the antibody or antigen-binding fragment through a lysine residue or a cysteine ​​residue (e.g., by cleavage of a disulfide group of the antibody or fragment, or by a modified cysteine ​​residue in the antibody or fragment). In some cases, the ligand is able to bind to the antibody or fragment through a glutamine residue, including those derived by transglutaminase-mediated conjugation.

[00295] Exemplary ligands that can be used in the context of the present invention include ligands comprising or consisting of, for example, MC (6-maleimidocaporyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alaninephenylalanine), phe-lys (phenylalanine-lysine), protease-cleavable ligand dipeptide site, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate), and variants and combinations thereof. Additional examples of ligands that can be used in the context of the present invention are disclosed, for example, in patent document no. 7,754,681 and in Ducry, Bioconjugated Chem., 2010, 21:5 to 13, and the references cited in this document, the content of which is incorporated by reference in this document in its entirety. In some cases, the ligand is or contains a self-immolating spacer, such as those discussed in Jin, et al., Bioorganic & Medicinal Chemistry, 2012, 20:3.465 to 3.469, and Wu, et al., Bioorganic & Medicinal Chemistry, 2016, 24:2.697 to 2.706.

[00296] Charges can be attached to the anti-MUC16 antibody or antigen-binding fragment by means of attachment to a particular amino acid within the antibody or antigen-binding molecule. Petition 870260046565, dated 05 / 15 / 2026, p. 129 / 435 121 / 202 antigen. Exemplary amino acid fixations that may be used in the context of the present aspect of the invention include, for example, lysine (see, for example, U.S. Patent No. 5,208,020; U.S. document 2010 / 0129314; Hollander et al., Bioconjugated Chem., 2008, 19:358 to 361; WO document 2005 / 089808; U.S. Patent No. 5,714,586; U.S. document 2013 / 0101546; and U.S. document 2012 / 0585592), cysteine ​​(see, for example, U.S. document 2007 / 0258987; WO document 2013 / 055993; WO document 2013 / 055990; WO document 2013 / 053873; WO document 2013 / 053872; document WO 2011 / 130598; al., Proc. Natl. Academic. Academic. 110:46 to 51, and Rabuka et al., Nat. Protocols, 2012, 10: 1052 to 1067), non-natural amino acids (see, for example, WO 2013 / 068874 and WO 2012 / 166559) and acidic amino acids (see, for example, WO 2012 / 05982). Ligands can also be conjugated to an antigen-binding protein by attachment to carbohydrates (see, for example, US 2008 / 0305497, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide ligands (see, for example, WO 2013 / 085925, WO 2010 / 010324, WO 2011 / 018611, and Shaunak et al., Nat. Chem.

[00297] The drug-to-antibody ratio (DAR) is the average number of drug conjugates to the antibody or antigen-binding fragment, which has a significant effect on the efficacy, potency, and pharmacokinetics of ADC. In various embodiments, the DAR is 1, 2, 3, 4, 5, 6, 7, or 8 drug molecules per antibody. In some Petition 870260046565, dated 05 / 15 / 2026, pp. 130 / 435 In 122 / 202 modalities, the DAR is 1 to 4. In certain modalities, the DAR is 2 to 4. In some cases, the DAR is 2 to 3. In certain cases, the DAR is 3 to 4. In some modalities, the DAR is 1 to 10, 1 to 20, or 1 to 30 (i.e., 1 to 30 drug molecules per antibody or antigen-binding fragment). THERAPEUTIC FORMULATION AND ADMINISTRATION

[00298] The present invention provides pharmaceutical compositions comprising the antigen-binding molecules of the present invention. The pharmaceutical compositions of the invention are formulated with vehicles, excipients, and other agents that provide enhanced transfer, delivery, tolerance, and the like. A variety of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gelatins, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbocera emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbocera. See also Powell et al.Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52:238 to 311.

[00299] The dose of antigen-binding molecule administered to a patient may vary depending on the patient's age and size, the target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When a bispecific antigen-binding molecule of the present invention is used for therapeutic purposes in an adult patient, it may be advantageous to administer the antigen-binding molecule intravenously. Petition 870260046565, dated 05 / 15 / 2026, page 131 / 435 The bispecific antigen-binding molecule of the present invention is typically administered in a single dose of about 0.01 to about 20 mg / kg of body weight, more 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 condition, the frequency and duration of treatment may be adjusted. Effective dosages and schedules for administering a bispecific antigen-binding molecule can be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Furthermore, interspecies dose escalation can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[00300] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, for example, 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. 262:4,429 to 4,432). Methods 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 epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents. Administration can be systemic or local.

[00301] A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard syringe or needle. Furthermore, with regard to subcutaneous delivery, a pen-based delivery device readily has applications in Petition 870260046565, dated 05 / 15 / 2026, page 132 / 435 124 / 202 delivery of a pharmaceutical composition of the present invention. Such a pen delivery device may be reusable or disposable. A reusable pen delivery device generally uses a replaceable cartridge containing a pharmaceutical composition. Once all the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is supplied pre-filled with the pharmaceutical composition retained in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[00302] Numerous pen-type and reusable autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, United Kingdom), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, 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 mention just a few.Examples of disposable pen delivery devices that have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly) autoinjector. Petition 870260046565, dated 05 / 15 / 2026, page 133 / 435 125 / 202 SURECLICK™ (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, LP), and the HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to mention just a few.

[00303] In certain situations, the pharmaceutical composition can be delivered in 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, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled-release system may be placed close to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pages 115 to 138). Other controlled-release systems are discussed in the review by Langer, 1990, Science 249:1527 to 1533.

[00304] Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared, for instance, by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As an aqueous medium for injections, there is, for example, physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent, such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a non-ionic surfactant [for Petition 870260046565, dated 05 / 15 / 2026, page 134 / 435 126 / 202 example, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)] etc. As an oily medium, sesame oil, soybean oil, etc., are employed, for example, which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into an appropriate ampoule.

[00305] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in single-dose dosage forms suitable for adjusting a dose of the active ingredients. Such single-dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally from about 5 to about 500 mg per single-dose dosage form; especially in the injection form, it is preferable that the antibody be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms. THERAPEUTIC USES OF ANTIGEN-BINDING MOLECULES

[00306] The present invention includes methods comprising administering to an individual in need thereof a therapeutic composition comprising an anti-MUC16 antibody or antigen-binding fragment thereof, or a bispecific antigen-binding molecule that binds specifically to CD3 and MUC16. The therapeutic composition may comprise any of the antibodies or bispecific antigen-binding molecules as disclosed herein and a pharmaceutically acceptable diluent or vehicle. As used herein, the expression "an individual in need thereof" means a human being or non-human animal exhibiting one or more symptoms or indications of cancer (e.g., an individual expressing a Petition 870260046565, dated 05 / 15 / 2026, page 135 / 435 127 / 202 tumor or suffering from any of the cancers mentioned in this document below), or who would otherwise benefit from an inhibition or reduction in MUC16 activity or a depletion of MUC16+ cells (e.g., ovarian cancer cells).

[00307] The antibodies and bispecific antigen-binding molecules of the invention (and therapeutic compositions comprising the same) are useful, among other things, for treating any disease or disorder in which the stimulation, activation and / or targeting of an immune response will be beneficial. In particular, the anti-MUC16 antibodies or the anti-CD3 / anti-MUC16 bispecific antigen-binding molecules of the present invention can be used for the treatment, prevention and / or improvement of any disease or disorder associated with or mediated by MUC16 expression or activity or proliferation of MUC16+ cells. The mechanism of action by which the therapeutic methods of the invention are achieved includes the extermination of MUC16-expressing cells in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis or by a combination of two or more of these mechanisms.Cells expressing MUC16 that can be inhibited or killed using the bispecific antigen-binding molecules of the invention include, for example, ovarian cancer cells.

[00308] The antigen-binding molecules of the present invention can be used to treat a disease or disorder associated with MUC16 expression, including, for example, cancer such as ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma mass-forming type, cervical adenocarcinoma, and gastric tract adenocarcinoma. According to certain embodiments of the present invention, anti-MUC16 antibodies or antibodies Petition 870260046565, dated 05 / 15 / 2026, page 136 / 435 128 / 202 bispecific anti-MUC16 / anti-CD3 antibodies are useful for treating a patient affected by ovarian cancer. According to other related embodiments of the invention, methods are provided comprising administering an anti-MUC16 antibody or a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule as disclosed herein to a patient affected by ovarian cancer. Analytical / diagnostic methods known in the art, such as tumor scanning, etc., can be used to determine if a patient has an ovarian tumor.

[00309] The present invention also includes methods for treating residual cancer in an individual. As used herein, the term residual cancer means the existence or persistence of one or more cancerous cells in an individual after treatment with an anticancer therapy.

[00310] In certain aspects, the present invention provides methods for treating a disease or disorder associated with MUC16 expression (e.g., ovarian cancer) comprising administering one or more of the anti-MUC16 or bispecific antigen-binding molecules described elsewhere in this document to an individual after it has been determined that the individual has prostate cancer. For example, the present invention includes methods for treating ovarian cancer comprising administering an anti-MUC16 antibody or a bispecific anti-CD3 / anti-MUC16 antigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more after the individual has received hormone therapy (e.g., antiandrogen therapy). COMBINATION THERAPIES AND FORMULATIONS

[00311] The present invention provides methods comprising administering a pharmaceutical composition comprising any Petition 870260046565, dated 05 / 15 / 2026, page 137 / 435 129 / 202 among the exemplary antibodies and bispecific antigen-binding molecules described in this document in combination with one or more additional therapeutic agents. Additional exemplary therapeutic agents that may be combined with or administered in combination with an antigen-binding molecule of the present invention include, for example, an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule EGFR inhibitor [e.g., gefitinib or erlotinib]), an antagonist of another member of the EGFR family such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., anti-ErbB2, anti-ErbB3, or anti-ErbB4 antibody or a small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), an EGFRvIII antagonist (e.g., an antibody that specifically binds to EGFRvIII), a cMET antagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody),a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody), a VEGF antagonist (e.g., a VEGF-Trap, see, for example, document no. US 7,087,411 (also referred to herein as a VEGF inhibition fusion protein)), an anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase receptor inhibitor (e.g., sunitinib, sorafenib, or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in document no. US 2009 / 0142354 as REGN421), an Ang2 antagonist (e.g., an antibody anti-Ang2 disclosed in document no. US 2011 / 0027286 as H1H685P), a FOLH1 (PSMA) antagonist, a PRLR antagonist (e.g., an anti-PRLR antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-Ang2 antibody, Petition 870260046565, dated 05 / 15 / 2026, pp. 138 / 435 130 / 202 STEAP1 or an anti-STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), an MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakine antagonist (e.g., an anti-uroplakine antibody), etc. Other agents that may be beneficially administered in combination with the antigen-binding molecules of the invention include cytokine inhibitors, including cytokine inhibitors and small molecule 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.The pharmaceutical compositions of the present invention (for example, pharmaceutical compositions comprising a bispecific antigen-binding molecule anti-CD3 / anti-MUC16 as disclosed herein) can also be administered as part of a therapeutic regimen comprising one or more therapeutic combinations selected from ICE: ifosfamide (e.g., Ifex®), carboplatin (e.g., Paraplatin®), etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16); DHAP: dexamethasone (e.g., Decadron®), cytarabine (e.g., Cytosar-U®, cytosine arabinoside, ara-C), cisplatin (e.g., Platinol®-AQ); and ESHAP: etoposide (e.g., Etopophos®, Toposar®, VePesid®, VP-16), methylprednisolone (e.g., Medrol®), high-dose cytarabine, cisplatin (e.g., Platinol®-AQ).

[00312] The present invention also includes therapeutic combinations comprising any of the antigen-binding molecules mentioned 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 (PSMA), PRLR, STEAP1, STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementioned cytokines, wherein the inhibitor is a Petition 870260046565, dated 05 / 15 / 2026, p. 139 / 435 131 / 202 aptamer, an antisense molecule, a ribozyme, an siRNA, a pepticobody, a nanobody or an antibody fragment (e.g., Fab fragment; F(ab')2 fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or other modified molecules such as diabodies, triabodies, tetrabodies, minibodies and minimal recognition units). The antigen-binding molecules of the invention can also be administered and / or co-formulated 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 conventional radiation and / or chemotherapy.

[00313] The additional therapeutically active component (or components) may be administered immediately before, simultaneously with, or shortly after the administration of an antigen-binding molecule of the present invention; (for the purposes of the present disclosure, such administration regimens are considered to be the administration of an antigen-binding molecule in combination with an additional therapeutically active component).

[00314] The present invention includes pharmaceutical compositions in which an antigen-binding molecule of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere in this document. ADMINISTRATIVE REGIMES

[00315] According to certain embodiments of the present invention, multiple doses of an antigen-binding molecule (for example, an anti-MUC16 antibody or a bispecific antigen-binding molecule that binds specifically to MUC16 and CD3) can be administered to an individual over a defined period of time. The methods according to the present aspect of the invention Petition 870260046565, dated 05 / 15 / 2026, p. 140 / 435 132 / 202 comprise sequentially administering to an individual multiple doses of an antigen-binding molecule of the invention. As used herein, sequential administration means that each dose of an antigen-binding molecule is administered to the individual at a different point in time, for example, on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods comprising sequentially administering to the patient a single initial dose of an antigen-binding molecule, followed by one or more secondary doses of the antigen-binding molecule, and optionally followed by one or more tertiary doses of the antigen-binding molecule.

[00316] The terms initial dose, secondary doses, and tertiary doses refer to the temporal sequence of administration of the antigen-binding molecule of the invention. Therefore, the initial dose is the dose administered at the beginning of the treatment regimen (also called the baseline dose); secondary doses are the doses administered after the initial dose; and tertiary doses are the doses administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of the antigen-binding molecule, but they may generally differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of an antigen-binding molecule contained in the initial, secondary, and / or tertiary doses varies from one to another (e.g., adjusted upwards or downwards as appropriate) during the course of treatment.In certain modalities, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of the treatment regimen as loading doses followed by subsequent doses that are administered on a smaller basis. Petition 870260046565, dated 05 / 15 / 2026, p. 141 / 435 133 / 202 frequent (e.g., maintenance doses).

[00317] In an exemplary embodiment of the present invention, each secondary and / or tertiary dose is administered from 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 22, 22½). The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of antigen-binding molecule that is administered to a patient before the administration of the immediately following dose, in a sequence without intervening doses.

[00318] Methods according to this aspect of the invention may comprise administering to a patient any number of secondary and / or tertiary doses of an antigen-binding molecule (for example, an anti-MUC16 antibody or a bispecific antigen-binding molecule that binds specifically to MUC16 and CD3). For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (for example, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (for example, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[00319] In modalities involving multiple secondary doses, each secondary dose may be administered with the same frequency as other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in modalities involving multiple tertiary doses, each tertiary dose may be Petition 870260046565, dated 05 / 15 / 2026, p. 142 / 435 134 / 202 is administered with the same frequency as other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and / or tertiary doses are administered to a patient may vary throughout the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the individual patient's needs after clinical examination. Diagnostic uses of antibodies

[00320] The anti-MUC16 antibodies of the present invention can also be used to detect and / or measure MUC16, or cells expressing MUC16 in a sample, for example, a biological sample for diagnostic purposes. For example, an anti-MUC16 antibody, or a fragment thereof, can be used to diagnose a condition or disease characterized by aberrant expression (e.g., overexpression, underexpression, lack of expression, etc.) of MUC16. Exemplary diagnostic assays for MUC16 may comprise, for example, placing a sample, obtained from a patient, in contact with an anti-MUC16 antibody of the invention, wherein the anti-MUC16 antibody is identified with a detectable identifier or reporter molecule. Alternatively, an unidentified anti-MUC16 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably identified.The detectable identifier or reporter molecule may be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I; a fluorescent or chemiluminescent chemical moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Another exemplary diagnostic use of the anti-MUC16 antibodies of the invention includes antibody identified as 89Zr, as identified as 89Zr. Petition 870260046565, dated 05 / 15 / 2026, page 143 / 435 135 / 202 deferoxamine, for the purpose of non-invasive identification and tracking of tumor cells in an individual (e.g., positron emission tomography (PET) imaging). (See, for example, Tavare, R. et al. Cancer Res. 2016 Jan 1;76(1):73-82; and Azad, BB. et al. Oncotarget. 2016 Mar 15;7(11):12344-58). Specific exemplary assays that can be used to detect or measure MUC16 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[00321] Samples that can be used in MUC16 diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient that contains detectable amounts of MUC16 protein, or fragments thereof, under normal or pathological conditions. Generally, MUC16 levels in a particular sample obtained from a healthy patient (e.g., a patient not affected by a disease or condition associated with abnormal MUC16 levels or activity) will be measured to initially establish a baseline, or standard, level of MUC16. This baseline level of MUC16 can then be compared against MUC16 levels measured in samples obtained from individuals suspected of having a disease or condition related to MUC16 (e.g., a tumor containing cells expressing MUC16).Examples of tissue or fluid samples include, but are not limited to, plasma, serum, ascites, ovary, uterus, cervix, liver, bladder, pancreas, stomach, small and large intestines, gallbladder, breast, lung, kidney, salivary and lacrimal glands, or any epithelioid malignancy thereof. Additional examples of tissue or fluid samples include, but are not limited to, papillary serous carcinoma of the cervix, endometrial adenocarcinoma, and adenocarcinoma of the uterine cell. Petition 870260046565, dated 05 / 15 / 2026, p. 144 / 435 136 / 202 clear bladder, seminal vesicle carcinoma, gastric carcinoma, colorectal adenocarcinoma, and epithelioid mesothelioma. It is contemplated that any fluid or tissue sample containing detectable amounts of MUC16 protein, or fragments thereof, may be subjected to the detection methods described herein. The methods described may be used to monitor the development and progression of malignancies, or to distinguish between normal and disease conditions. Thus, the methods described may be used to detect or monitor cancers such as ovarian cancer, bladder cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma mass-forming type, cervical adenocarcinoma, and gastric tract adenocarcinoma. EXAMPLES

[00322] The following examples are presented so as to provide those of ordinary skill in the art with a full disclosure and description of how to produce and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperature, etc.), however, some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. EXAMPLE 1: GENERATION OF ANTI-MUC16 ANTIBODIES

[00323] Anti-MUC16 antibodies were obtained by immunizing a genetically modified mouse with a human MUC16 antigen or by immunizing a modified mouse comprising DNA encoding variable kappa light and heavy chain regions of Petition 870260046565, dated 05 / 15 / 2026, page 145 / 435 137 / 202 human immunoglobulin with a human MUC16 antigen.

[00324] Genetically modified mice were immunized with hMUC16.nub (a truncated form encompassing the last five SEA domains of Mucin-16 (SEQ ID: 1902)), or immunized with a cell line expressing hMUC16, such as OVCAR-3 type cells. SEQ ID NO: 1902 contains residues 13,810 to 14,451 of SEQ ID NO: 1899, as well as C-terminal tags. After immunization, splenocytes were collected from each mouse and (1) fused with mouse myeloma cells to preserve their viability and form hybridoma cells and examined for MUC16 specificity, or (2) sorted by B cell (as described in US document 2007 / 0280945A1) using a fragment of human MUC16 as the sorting reagent that binds to and identifies reactive antibodies (antigen-positive B cells).

[00325] Chimeric antibodies to MUC16 were initially isolated having a human variable region and a mouse constant region. The antibodies were characterized and selected for desirable characteristics, including affinity, selectivity, etc. If necessary, the mouse constant regions were replaced with a desired human constant region, for example, a wild-type or modified IgG1 or IgG4 constant region, to generate a fully human anti-MUC16 antibody. Although the selected constant region may vary according to the specific use, high-affinity antigen binding and target specificity characteristics are located in the variable region. Antibody name designations such as H1H8755P and H1M7129N denote fully human H1H antibodies or chimeric mouse constant region / human variable region H1M antibodies. Antibodies identified by the hybridoma method are indicated with Petition 870260046565, dated 05 / 15 / 2026, page 146 / 435 138 / 202 antibody ID numbers ending with N or N2. Antibodies identified by the B-cell sorting method are indicated with antibody ID numbers ending in P or P2.

[00326] Certain biological properties of the exemplary anti-MUC16 antibodies generated according to the methods of this Example are described in detail in the Examples presented below. AMINO ACID AND NUCLEIC ACID SEQUENCES OF THE VARIABLE REGION OF HEAVY AND LIGHT CHAINS OF ANTI-MUC16 ANTIBODIES

[00327] Table 1 presents the amino acid sequence identifiers of the variable heavy and light chain regions and CDRs of selected anti-MUC16 antibodies from the invention. The corresponding nucleic acid sequence identifiers are presented in Table 2. TABLE 1: AMINO ACID SEQUENCE IDENTIFIERS SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H8755P 2 4 6 8 10 12 14 16 H1H8767P 18 20 22 24 26 28 30 32 H1H8770P 34 36 38 40 42 44 46 48 H1H8783P 50 52 54 56 58 60 62 64 H1H8790P 66 68 70 72 74 76 78 80 H1H8794P 82 84 86 88 90 92 94 96 H1H8794P2 82 84 86 88 858 860 862 864 H1H8799P 98 100 102 104 106 108 110 112 H1H8799P2 98 100 102 104 170 172 174 176 H1H8804P 114 116 118 120 122 124 126 128 H1H8808P 130 132 134 136 138 140 142 144 H1H8810P 146 148 150 152 154 156 158 160 H1H8813P 162 164 166 168 170 172 174 176 Petition 870260046565, dated 05 / 15 / 2026, page 147 / 435 139 / 202 H1M7129N 178 180 182 184 186 188 190 192 216 H1M9521N 218 220 222 224 226 228 230 232 H1M9528N 234 236 238 240 242 244 246 248 H2M7128N 250 252 254 256 1936 1938 1940 1942 H1M7130N 1944 1946 1948 1950 1952 1954 1956 1958 H2M7131N 258 260 262 264 266 268 270 272 H2M7133N 274 276 278 280 1936 1938 1940 1942 H2M7134N 282 284 286 288 290 292 294 296 H2M7135N 298 300 302 304 306 308 310 312 H2M7138N 314 316 318 320 322 324 Table 2: Acid Sequence Identifiers NUCLEAR SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H8755P 1 3 5 7 9 11 13 15 H1H8767P 17 19 21 23 25 27 29 31 H1H8770P 33 35 37 39 41 43 45 47 H1H8783P 49 51 53 55 57 59 61 63 H1H8790P 65 67 69 71 73 75 77 79 H1H8794P 81 83 85 87 89 91 93 95 H1H8794P2 81 83 85 87 857 859 861 863 H1H8799P 97 99 101 103 105 107 109 111 H1H8799P2 97 99 101 103 169 171 173 175 H1H8804P 113 115 117 119 121 123 125 127 H1H8808P 129 131 133 135 137 139 141 143 Petition 870260046565, dated 05 / 15 / 2026, page 148 / 435 140 / 202 H1H8810P 145 147 149 151 153 155 157 159 191 H1M7137N 193 195 197 199 393 395 397 399 H1M9519N 201 203 205 207 209 211 213 215 H1M9521N 217 219 221 223 225 227 229 231 H1M9528N 233 235 237 239 241 243 245 247 H2M7128N 249 251 253 255 1935 1937 1939 1941 1953 1955 1957 H2M7131N 257 259 261 263 265 267 269 271 H2M7133N 273 275 277 279 1935 1937 1939 1941 H2M7134N 281 283 285 287 289 291 293 295 H2M7135N 297 299 301 303 305 307 309 311 H2M7138N 313 315 317 319 321 323 325 327 H2M9538N 329 331 333 335 337 339 341 343 H3M9524N 345 ​​347 349 351 353 355 357 359 H3M9525N 361 363 365 367 369 371 373 375 H3M9529N 377 379 381 383 385 387 389 391 EXAMPLE 2: ANTI-MUC16 ANTIBODIES BIND Specifically, hMUC16 is endogenously expressed in the OVCAR-3 cell line.

[00328] The ability of anti-MUC16 antibodies to specifically bind to endogenously expressed MUC16 in the human ovarian carcinoma cell line (OVCAR-3) was evaluated using an electrochemiluminescence-based detection assay (Meso Scale Discovery (MSD), Rockville, MD). Briefly, OVCAR-3 and a control ovarian adenocarcinoma cell line, SK-OV-3, which does not have detectable hMUC16 expression, were rinsed in 1xPBS supplemented with Ca2+ / Mg2+ (Irvine Scientific, Santa Ana, CA) followed by incubation in Enzyme-Free Cell Dissociation Buffer (Millipore, Billerica, MA) for 10 min at 37 °C. The separated cells were then washed once in 1xPBS supplemented Petition 870260046565, dated 05 / 15 / 2026, page 149 / 435 141 / 202 cells with Ca2+ / Mg2+ and counted (Cellometer Auto T4 cell counter, Nexcelom Bioscience, Lawrence, MA). Approximately 1.0 x 10⁴ cells were arranged in 96-well MULTI-ARRAY (MSD) carbon electrode plates and incubated for 1 h at 37°C. Non-specific binding sites were then blocked with 2% BSA (w / v) in PBS for 1 h at room temperature. Subsequently, serial dilutions of anti-MUC16 or control antibodies (0.85 pM to 50 nM) and no antibody buffer were added to the plated cells and incubated for 1 h at room temperature (RT). The plates were then washed to remove unbound antibodies using an AquaMax2000 plate washer (MDS Analytical Technologies, Sunnyvale, CA). Antibodies arranged on plates were detected with a SULFO-TAG™-conjugated anti-human kappa light chain antibody (Regeneron) or a SULFO-TAG™-conjugated anti-mouse IgG antibody (Jackson Immunoresearch, West Grove, PA) for 1 h at RT.After washing, the plates were developed with Reading Buffer (MSD) according to the manufacturer's protocol and the luminescent signals were recorded with a SECTOR Imager 6000 (MSD) instrument.

[00329] The luminescence intensity, measured in relative light units (RLU), for the two cell lines was recorded to indicate the binding intensity of each antibody. The signal ratio detected with 1.9 nM or 16.7 nM anti-MUC16 antibody binding to OVCAR-3 vs. SK-OV-3 was reported as an indication of specificity and binding potency (Table 3). TABLE 3: CELLULAR BINDING RATIOS OF ANTIMUC16 ANTIBODIES TO ENDOGENOUSLY EXPRESSING hMUC16c OVCAR-3 VERSUS dhMUC16-NEGATIVE SK-OV-3 CELL LINES Petition 870260046565, dated 05 / 15 / 2026, page 150 / 435 142 / 202 Reason: OVCAR-3 / SK-OV-3 Anti-MUC16 Binding Antibody ID [Ab]: 1.9nM [Ab] 16.7nM Hybridoma Anti-Muc16 Antibodies (HIM, H2M, H3M) H2M7128N 59 31 H1M7129N 68 19 H2M7131N 86 37 H2M7133N 69 33 H2M7134N 68 29 H2M7135N 30 5 H1M7137N 77 29 H2M7138N 82 50 H3M7132N 89 38 H1M9519N 109 78 H1M9521N 132 107 H3M9524N 81 57 H3M9525N 137 51 H1M9528N 153 120 H3M9529N 143 99 H2M9538N 89 26 Anti-Human Fc MUC16 Antibodies (H1H) H1H8755P 13 5 H1H8767P 4 NS H1H8770P 9 3 H1H8783P 11 4 H1H8790P 8 3 Petition 870260046565, dated 05 / 15 / 2026, page 151 / 435 143 / 202 Reason: OVCAR-3 / SK-OV-3 Binding of antiMUC16 Antibody ID [Ab]: 1.9nM [Ab]: 16.7nM H1H8794P 8 3 H1H8794P2 5 NS H1H8799P 10 4 H1H8799P2 10 4 H1H8804P 8 3 H1H8808P 4 NS H1H8810P 8 3 H1H8813P 11 5 H1H9519N 41 30 H1H9521N 30 28 H1H9524N 20 48 H1H9525N 4 21 H1H9528N 41 18 H1H9529N 109 74 H1H9538N 12 13 Controls Isotope Control Antibody of mIgG1 NS NS Isotope Control Antibody of mIgG2a NS NS Isotope Control Antibody of hIgG1 NS NS NS = Non-Specific - ratio at 1.9 nM or 16.7 nM < 2.5 times. Isotypes: H1H: hIgG1; H1M: mIgG1; H2M: mIgG2; H3M: mIgG3 Petition 870260046565, dated 05 / 15 / 2026, p. 152 / 435 144 / 202 Note: Variation in the intensity of binding ratios between the same antibody expressed with a mouse Fc and a human Fc is due to the use of different SULFO-TAG secondary detection reagents.

[00330] As the results in Table 3 show, a majority of the anti-MUC16 antibodies of this invention bind specifically to OVCAR-3 at both high (16.7 nM) and low (1.9 nM) antibody concentrations. The mIgG1, mIgG2a, and hIgG1 Isotype Control antibodies did not show any specific binding to OVCAR-3 or the SK-OV-3 cell line. Additionally, there is evidence that the method is sensitive since several antibodies that did not exhibit binding to soluble monomeric human MUC16 protein in a surface plasmon resonance binding assay (see Example 4 hereafter) exhibited specific binding to endogenous human MUC16 expressed in OVCAR-3 type cells in this cell-based binding assay. EXAMPLE 3: GENERATION OF BIESPECIFIC ANTIBODIES THAT BIND TO (MUC16) AND OVARIAN CELL-SPECIFIC CD3

[00331] The present invention provides bispecific antigen-binding molecules that bind to CD3 and MUC16; such bispecific antigen-binding molecules are also referred to herein as anti-MUC16 / anti-CD3 or antiMUC16xCD3 bispecific molecules. The anti-MUC16 portion of the bispecific antiMUC16 / anti-CD3 molecule is useful for targeting tumor cells that express MUC16 (also known as CA-125), and the anti-CD3 portion of the bispecific molecule is useful for activating T cells. The simultaneous binding of MUC16 to a tumor cell and CD3 to a T cell facilitates the targeted killing (cell lysis) of a tumor cell by the activated T cell.

[00332] Bispecific antibodies comprising a domain Petition 870260046565, dated 05 / 15 / 2026, p. 153 / 435 145 / 202 specific anti-MUC16 binding domains and an anti-CD3 specific binding domain were constructed using standard methodologies, wherein the anti-MUC16 antigen-binding domain and the anti-CD3 antigen-binding domain each comprise different distinct HCVRs paired with a common LCVR. In exemplified bispecific antibodies, molecules were constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a common light chain from the anti-MUC16 antibody. In other cases, bispecific antibodies may be constructed using a heavy chain from an anti-CD3 antibody, a heavy chain from an anti-MUC16 antibody, and a light chain from an anti-CD3 antibody, or a light chain from an antibody known to be promiscuous or to pair effectively with a variety of heavy chain arms.The bispecific antibodies described in the following examples consist of anti-CD3 binding arms that have varying binding affinities for human soluble heterodimeric hCD38 / Q protein (as described in Example 15 in this document); and human MUC16 (see Examples 1 to 2 above). The exemplified bispecific antibodies were manufactured having either an IgG1 Fc domain (BSMUC16 / CD3-001, -002, -003 and -004) or a modified (chimeric) IgG4 Fc domain (BSMUC16 / CD3-005) as disclosed in U.S. Patent Application Publication No. 20140243504A1, published August 28, 2014.

[00333] A summary of the component parts of the antigen-binding domains of the various constructed antiMUC16xCD3 bispecific antibodies is shown in Table 4. TABLE 4: SUMMARY OF COMPONENT PARTS OF Selected bispecific anti-MUC16xCD3 antibodies Petition 870260046565, dated 05 / 15 / 2026, p. 154 / 435 146 / 202 Bispecific Antibody Identifier Anti-MUC16 Anti-CD3 Common Light Chain Variable Region Antigen-Binding Domain Antigen-Binding Domain Heavy Chain Variable Region Heavy Chain Variable Region BSMUC16 / CD3-001 H1H8767P (SEQ ID NO:18) CD3-VH-G (SEQ ID NO:1730) H1H8767P (SEQ ID NO: 26) BSMUC16 / CD3-002 H1H8767P (SEQ ID NO:18) CD3-VH-G5 (SEQ ID NO:1762) H1H8767P (SEQ ID NO: 26) BSMUC16 / CD3-003 H1H8767P (SEQ ID NO:18) CD3-VH-G9 (SEQ ID NO:1778) H1H8767P (SEQ ID NO: 26) BSMUC16 / CD3-004 H1H8767P (SEQ ID NO:18) CD3-VH-G10 (SEQ ID NO:1786) H1H8767P (SEQ ID NO: 26) BSMUC16 / CD3-005 H1H8767P (SEQ ID NO:18) CD3-VH-G20 (SEQ ID NO:1866) H1H8767P (SEQ ID NO: 26)

[00334] The light chains listed in Table 4 were common to the targeting arms of both CD3 and MUC16 of the bispecific antibodies. Tables 1 and 2 present amino acid and nucleic acid sequence identifiers, respectively, for the various variable heavy chain regions, and their corresponding CDRs, of the anti-MUC16 arms of the bispecific antibodies in this Example. Tables 22 and 23 present amino acid and nucleic acid sequence identifiers, respectively, for the various variable heavy chain regions, and their corresponding CDRs, of the anti-CD3 arms of the bispecific antibodies in this Example. EXAMPLE 4: BINDING AFFINITIES AND SURFACE PLASMON RESONANCE KINETIC CONSTANTS DERIVED FROM HUMAN MONOCLONAL ANTIBODIES Petition 870260046565, dated 05 / 15 / 2026, page 155 / 435 147 / 202 MONOSPECIFIC ANTI-MUC16 AND BIESPECIFIC ANTIMUC16xCD3

[00335] The binding affinities and kinetic constants of human anti-MUC16 antibodies were determined by real-time surface plasmon resonance (SPR; Biacore 4000 or Biacore T-200, GE Healthcare Life Sciences, Pittsburgh, PA) at 25°C. The anti-MUC16 antibodies tested in this example were bivalent monospecific agglutinators to MUC16 (expressed with a constant region of hIgG1 (H1H), mIgG1 (H1M), mIgG2 (H2M), or mIgG3 (H3M)) or bispecific antibodies comprising an anti-MUC16 binding domain and an anti-CD3 binding domain. Antibodies were captured on a CM4 or CM5 Biacore sensor surface (GE Healthcare Life Sciences) derived via amine coupling with a human anti-Fc monoclonal antibody (GE, # BR-1008-39) or a goat mouse anti-Fc monoclonal antibody (GE, # BR-1008-38).Various concentrations of soluble monomeric human MUC16, in a truncated form encompassing the last five SEA domains of Mucin-16 (hMUC16.mmh, or MUC16 nub, SEQ ID: 1902), were injected onto the captured surface of anti-MUC16 antibody at a flow rate of 50 pL / minute (Biacore T-200) or 30 pL / minute (Biacore 4000). Antibody-reagent binding was monitored for 4 min and dissociation was monitored for 6 to 10 min. All binding studies were performed in HBS-ET buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.05% v / v P20 Surfactant).

[00336] The kinetic association (ka) and dissociation rate constants (kd) were determined by fitting the sensograms in real time to a 1:1 linkage model using Scrubber 2.0c curve fitting software. The linkage dissociation equilibrium constants (Kd) and dissociative half-lives (0:½) were Petition 870260046565, dated 05 / 15 / 2026, page 156 / 435 148 / 202 calculated from the kinetic rate constants as: ln(2) kd t' / 2(min) = ----7-7 Kd (M) = —, e60 kd

[00337] The binding kinetics parameters for monospecific anti-MUC16 antibodies to a monomeric human MUC16 fragment protein are shown below in Tables 5A and 5B. The binding kinetics parameters for bispecific anti-MUC16 / anti-CD3 antibodies to monomeric human MUC16 protein are shown below in Table 6. TABLE 5A: BIACORE BINDING AFFINITIES OF HYBRIDOMA ANTI-MUC16 ANTIBODIES (H1M, H2M, AND H3M) TO THE hMUC16 FRAGMENT AT 25 °C Antibody ID ka (1 / Ms) kd (1 / s) KD (M) 11 / 2 (min) H2M7128N 1.89E+05 6.40E-04 3.39E-09 18 H1M7129N 5.31 E+04 1.04E-04 1.97E-09 111 H2M7131N 6.47E+04 1.62E-04 2.51 E-09 71 H3M7132N 2.57E+04 1.96E-04 7.62E-09 59 H2M7133N 1.67E+05 3.77E-04 2.26E-09 31 H2M7134N 6.55E+04 1.62E-04 2.47E-09 71 H2M7135N 5.10E+04 2.18E-04 4.27E-09 53 H1M7137N 5.30E+04 9.09E-05 1.72E-09 127 H2M7138N 7.41 E+04 9.25E-05 1.25E-09 125 H1M9519N NB NB NB NB H1M9521N NB NB NB NB H3M9524N NB NB NB NB H3M9525N NB NB NB NB H1M9528N NB NB NB NB Petition 870260046565, dated 05 / 15 / 2026, page 157 / 435 149 / 202 Antibody ID ka (1 / Ms) kd (1 / s) KD (M) t 1 / 2 (min) H3M9529N NB NB NB NB Note: No connection TABLE 5B: BIACORE BINDING AFFINITIES OF HUMAN Fc ANTI-MUC16 ANTIBODIES (H1H) TO THE hMUC16 FRAGMENT AT 25°C Antibody ID ka (1 / Ms) kd (1 / s) KD (M) 11 / 2 (min) H1H8755P 5.22E+05 1.49E-04 2.86E-10 77 H1H8767P 1.17E+05 4.18E-04 3.58E-09 28 H1H8770P 2.47E+05 3.08E-04 1.25E-09 38 H1H8783P 1.74E+05 1.07E-04 6.14E-10 108 H1H8790P 1.01E+05 7.61E-04 7.53E-09 15 H1H8794P 3.62E+05 2.79E-04 7.71E-10 41 H1H8799P 7.90E+04 3.66E-04 4.63E-09 32 H1H8799P2 7.58E+04 3.73E-04 4.92E-09 31 H1H8804P 4.94E+04 6.07E-04 1.23E-08 19 H1H8808P 4.12E+03 2.16E-04 5.24E-08 54 H1H8810P 5.77E+04 3.16E-04 5.48E-09 37 H1H8813P 5.32E+04 2.32E-04 4.35E-09 50 TABLE 6: BIACORE BINDING UNITS Bispecific Anti-Muc16 / Anti-CD3 Antibodies FRAGMENT OF hMUC16 AT 25° C Bispecific Antibody Identifier ka (1 / Ms) kd (1 / s) KD (M) t 1 / 2 (min) BSMUC16 / CD3-001 9.48E+04 5.86E-04 6.18E-09 20 Petition 870260046565, dated 05 / 15 / 2026, page 158 / 435 150 / 202 BSMUC16 / CD3-005 9.41E+04 5.64E-04 6.00E-09 21

[00338] As the results show, a majority of the anti-MUC16 antibodies of the present invention are bound to soluble human MUC16 protein, some exhibiting subnanomolar affinity. Several antibodies (H1M9519N, H1M9521N, H3M9524N, H3M9525N, H1M9528N, H3M9529N) did not exhibit binding to the truncated form encompassing the last five SEA domains via surface plasmon resonance; however, they did exhibit specific binding to endogenous human MUC16 expressed in OVCAR-3 type cells in a cell-based binding assay. The bispecific Anti-MUC16xCD3 antibodies of the present invention also bound to soluble truncated human MUC16 protein, exhibiting nanomolar affinity in this assay. EXAMPLE 5: ADDITIONAL BINDING, CELL ACTIVATION, AND CYTOTOXICITY PROPERTIES OF BIOSPECIFIC ANTIBODIES EXEMPLIFIED

[00339] In this example, the ability of bispecific MUC16xCD3 antibodies to bind to cell lines expressing human CD3 (human T cell), compared to binding to specific target cell lines (specific to MUC16), by means of FACS was determined. Additionally, the ability of these bispecific antibodies to activate specific target cell lines (specific to MUC16) was also compared in a similar assay. TITRATION OF BIESPECIFIC ANTIBODY BINDING EXEMPLIFIED AS MEASURED BY FACS ANALYSIS

[00340] A. Briefly, flow cytometric analysis (i.e., fluorescence-activated cell sorting, or FACS) was used to determine the binding of bispecific antibodies to Jurkat cells or cells expressing human MUC16, followed by detection with an anti-human IgG antibody identified by Petition 870260046565, dated 05 / 15 / 2026, p. 159 / 435 151 / 202 phycoerythrin (PE) or identified by APC. Briefly, 2 x 10⁵ cells / well were incubated for 30 minutes at 4 °C with a serial dilution ranging from 1.33E-0.7M to 8.03E-1.2M of each test antibody or isotype control (antibody of the same isotype that binds to a different antigen without cross-reactivity to MUC16 or CD3). After incubation, the cells were washed twice with ice-cold PBS containing 1% filtered FBS, and a PE-conjugated or APC-conjugated antihuman secondary antibody was added to the cells and incubated for an additional 30 minutes. Wells containing no antibody or only secondary antibodies were also used as controls. After incubation, the cells were washed, resuspended in 200 μL of ice-cold PBS containing 1% filtered FBS, and analyzed by flow cytometry on a BD FACS Canto II. See Table 7A.

[00341] B. In separate experiments with conditions analogous to that described above, flow cytometric analysis (or FACS) was used to determine the binding of selected bispecific antibodies to Jurkat, PEO-1, OVCAR3-Luc, and cynomolgus T cells. For the titration analysis, serial dilutions of selected MUC16xCD3 bispecific antibodies were performed, a first isotype control antibody (a chimeric human IgG4 antibody that binds to an irrelevant human antigen without cross-reactivity to cynomolgus or human CD3) and a second isotype control antibody (a chimeric human IgG4 antibody that binds to an irrelevant human antigen without cross-reactivity to human MUC16), ranging from 66.6 nM to 0.001 nM. See Table 7B and Figures 11A to 11C.

[00342] For FACS analysis, cells were keyed by direct diffusion height versus direct diffusion area for single event selection, followed by lateral and direct diffusion. The EC50 for Petition 870260046565, dated 05 / 15 / 2026, p. 160 / 435 Cell binding titer 152 / 202 was determined using PRISM™ software (GraphPad Software, Inc., La Jolla, CA). Values ​​were calculated using 4-parameter nonlinear regression analysis (Liu, J., et al. 2005, Biotechnol Letters 27: 1821-1827). The EC50 value represents the concentration of the tested antibody at which 50% of its maximum binding is observed. TABLE 7A: FACS BINDING TO SPECIFIC CD3 AND MUC16 CELL LINES Bispecific Antibody Identifier Binding Arm Anti-CD3 FACS Binding Titration EC50 [M] Jurkat OVCAR3 (MUC16+) BSMUC16 / CD3-001 CD3-VH-G 3.21E-09 1.20E-09 BSMUC16 / CD3-002 CD3-VH-G5 Very weak 2.69E-09 TABLE 7B: FACS BINDING TO SPECIFIC CD3 AND MUC16 CELL LINES Bispecific Antibody Identifier Binding Arm Anti-CD3 FACS Binding Titration EC50 [M] PEO-1 (MUC16+) OVCAR3Luc (MUC16+) Jurkat Cynomolg T Cells BSMUC16 / CD3-001 CD3-VH-G 3.02E-09 1.32E-09 6.44E-09 1.56E-08 BSMUC16 / CD3-002 CD3-VH-G5 3.13E-09 Not tested 3.01E-07 No connection BSMUC16 / CD3-005 CD3-VH-G20 2.63E-09 1.47E-09 6.26E-08 1.17E-06

[00343] As shown in Tables 7A and 7B, the CD3-binding arms of each MUC16 bispecific antibody exhibited a cell-binding range for T cells expressing human CD3 and Petition 870260046565, dated 05 / 15 / 2026, p. 161 / 435 153 / 202 monkey (e.g., from 3.2 nM EC50 to very weak binding). The BSMUC16 / CD3-001 bispecific antibody showed a high measurement of binding to CD3-expressing cells (i.e., < 7 nM) while the BSMUC16 / CD3-002 bispecific antibody showed weak binding to no binding to human and monkey CD3-expressing cells. Unmeasurable binding, or no measurable binding, in the FACS assay or equivalent assay refers to a binding interaction between the antibody and its target antigen that is beyond the detection limit of the assay (e.g., at 1 pM or above).The tested bispecific antibodies exhibited similar cell binding in cell lines expressing MUC16, confirming that bispecific pairing with individual CD3 arms exhibiting high or weak (or unmeasurable) interactions with CD3 did not affect or diminish tumor-specific binding (MUC16-specific binding was less than or equal to 3 nM (high binding) in these examples). The first control antibody did not bind to CD3+ cells, and the second control antibody did not bind to MUC16+ cells. See also Figures 11A to 11C.

[00344] Antibodies that exhibit weak to undetectable binding to human CD3 are still considered advantageous for avidity-driven bispecific pairing, and have been further tested for cytotoxicity in in vitro (see below) and in vivo (Example 8) assays. T cell activation and tumor-specific cytotoxicity exhibited by bispecific antibodies as measured in vitro.

[00345] A. The specific killing of MUC16-expressing tumor target cells in the presence of CD3-based bispecific antibodies was monitored by flow cytometry. As reported Petition 870260046565, dated 05 / 15 / 2026, page 162 / 435 Previously, bispecific antibodies exhibited differential binding abilities to CD3 protein and CD3-expressing cell lines (i.e., very weak or strong binding). These same bispecific antibodies were tested for their ability to induce pure human T cells to redirect killing to target expression cells.

[00346] Briefly, cell lines expressing MUC16 (OVCAR3) were identified with 1 μM of the fluorescent tracking dye Violet Cell Tracker. After identification, the cells were plated overnight at 37 °C. Separately, human PBMCs were plated in RPMI media supplemented at 1x10⁶ cells / mL and incubated overnight at 37 °C to enrich lymphocytes, depleting adherent macrophages, dendritic cells, and some monocytes. The following day, target cells were co-incubated with unstimulated, adherent cell-depleted PBMCs (effector / target cell ratio of 4:1) and a serial dilution of relevant bispecific antibodies or isotype control (concentration range: 66.7 nM to 0.25 pM) for 48 hours at 37 °C. Cells were removed from cell culture plates using an enzyme-free cell dissociation buffer and analyzed by FACS.Consult the results shown in Table 8A.

[00347] B. In analogous studies, cell lines expressing MUC16 (PEO-1 or OVCAR3-Luc) were identified, placed on plates, and incubated overnight as described. Serial dilutions of bispecific MUC16xCD3 antibodies or isotype control were co-incubated. See the results shown in Tables 8B and 8C, and Figures 12A to 12B.

[00348] For FACS analysis, cells were labeled with a far-red dead / alive cell marker (Invitrogen). 5x10⁵ counting microspheres were added to each well. Petition 870260046565, dated 05 / 15 / 2026, page 163 / 435 155 / 202 immediately before FACS analysis. 1x10⁴ microspheres were collected for each sample. For the evaluation of extermination specificity, cells were keyed into live populations identified with Violet. The percentage of live population was recorded and used for the normalized survival calculation.

[00349] T cell activation was assessed by incubating cells with antibodies directly conjugated to CD2, CD69, and / or CD25, and reporting the percentage of early-activated T cells (CD69+) and / or late-activated T cells (CD25+) among the total T cells (CD2+). As the results in Tables 8A to 8C show, depletion of MUC16-expressing cells was observed with bispecific anti-MUC16xCD3 antibodies. All bispecific antibodies tested activated and directed human T cells to deplete target cells with picomolar EC5s. Additionally, the observed target cell lysis (depletion) was associated with an upregulation of CD69 (or CD25) in CD2+ T cells, also with picomolar (pM) EC5s.

[00350] Above all, the results of this example also demonstrate that a bispecific antibody constructed with a CD3-binding arm that exhibited weak to unmeasurable binding to the CD3 protein or CD3-expressing cells (i.e., CD3-VH-G5) still retained the ability to activate T cells and exhibited potent cytotoxicity of antigen-expressing tumor cells. TABLE 8A: CYTOTOXICITY AND T-CELL ACTIVATION PROPERTIES OF SELECTED MUC16xCD3 BIE-SPECIFIC ANTIBODIES Petition 870260046565, dated 05 / 15 / 2026, page 164 / 435 156 / 202 Bispecific Antibody Identifier Binding Arm Anti-CD3 OVCAR3 EC50 [M] of cell depletion EC50 [M] of T cell activation (upregulation of CD69) BSMUC16 / CD3-001 CD3-VH-G 2.24E-11 5.88E-12 BSMUC16 / CD3-002 CD3-VH-G5 3.06E-11 1.01E-11 TABLE 8B: CYTOTOXICITY AND ACTIVATION PROPERTIES MUC16xCD3 BIESPECIFIC ANTIBODIES T CELL SELECTED Bispecific Antibody Identifier EC50 [M] of PEO-1 cell depletion EC50 [M] of T cell activation (upregulation of CD69) EC50 [M] of T cell activation (upregulation of CD25) BSMUC16 / CD3-001 2.56E-11 8.34E-12 3.90E-11 BSMUC16 / CD3-002 6.75E-11 1.34E-11 8.89E-11 BSMUC16 / CD3-005 7.74E-11 1.72E-11 1.06E-10 TABLE 8C: CYTOTOXICITY AND T CELL ACTIVATION PROPERTIES OF SELECTED MUC16xCD3 BIESPECIFIC ANTIBODIES Bispecific Antibody Identifier EC50 [M] Depletion of OVCAR3-Luc Cells EC50 [M] T-cell activation (CD69 upregulation) EC50 [M] T-cell activation (CD25 upregulation) BSMUC16 / CD3-001 1.54E-11 2.98E-12 3.06E-11 BSMUC16 / CD3-005 5.16E-11 1.54E-11 1.17E-10 EXAMPLE 6: HYDROGEN / DEUTERIUM (H / D) EXCHANGE BASED ON Petition 870260046565, dated 05 / 15 / 2026, page 165 / 435 157 / 202 NO Epitope Mapping of Anti-Muc16 Antibodies H4sH8767P, H1H8794P2 and H1H8799P2 Binding to a Portion of the C-Term Domain of hMUC16

[00351] Experiments were conducted to determine the amino acid residues of MUC16 within the five C-terminal SEA domains (SEQ ID No:1902, hereinafter referred to as hMUC16.nub), with which the anti-MUC16 antibodies H4sH8767P, H1H8794P2 and H1H8799P2 interact. For this purpose, Hydrogen / Deuterium (H / D) exchange epitope mapping with mass spectrometry (HDX-MS) was used. A general description of the H / D exchange method is presented in Ehring (1999) Analytical Biochemistry 267(2):252 to 259; and Engen and Smith (2001) Anal. Chem. 73:256A to 265A.

[00352] The HDX-MS experiments were performed on an integrated Waters HDX / MS platform, consisting of a Leaptec HDX PAL system for deuterium identification, a Waters Acquity M-Class (auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (solvent manager μBϊηάπο) for the analytical column gradient, and a Synapt G2-Si mass spectrometer for peptide mass measurement.

[00353] The identification solution was prepared in 10 mM PBS buffer at D2O in pD 7.0. For deuterium identification, 3.8 μL of hMUC16.nub (12 pmol^L) or hMUC16.nub premixed with anti-MUC16 antibody H4sH8767P, H1H8794P2, or H1H8799P2 in a 2:1 molar ratio was incubated with 56.2 μL of the D2O identification solution for various time points (e.g., non-deuterated control = 0 s; deuterium identification: 1 min and 20 min). Deuteration was achieved by rapidly transferring 50 μL of sample to 50 μL of pre-cooled rapid-cooling buffer (0.2 M). Petition 870260046565, dated 05 / 15 / 2026, page 166 / 435 158 / 202 TCEP, 6 M guanidine chloride in 100 mM phosphate buffer, pH 2.5) and the mixed sample was incubated at 1.0 °C for two minutes. The rapidly cooled sample was then injected into a Waters HDX Manager for online pepsin / protease XIII digestion. The digested peptides were captured on an ACQUITY UPLC BEH C18 1.7-pm, 2.1 x 5 mm VanGuard pre-column at 0 °C and eluted onto an ACQUITY UPLC BEH C18 analytical column (1.7-pm, 1.0 x 50 mm) for a 9-minute gradient separation from 5% to 40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer used a cone voltage of 37 V, a scan time of 0.5 s, and a mass / charge range of 50 to 1,700 Th.

[00354] To identify the hMUC16.nub peptide residues with which H4sH8767P, H1H8794P2, and H1H8799P2 interact, CL-MS E data from the non-deuterated sample were processed and compared with a database that included sequences for hMUC16.nub, pepsin, and a random sequence using Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported into DynamX software and filtered by two criteria: 1) minimum products per amino acid: 0.25 and 2) replication file threshold: 2. DynamX software automatically determined the deuterium uptake of each peptide based on retention time and high mass accuracy (<10 ppm) at multiple time points with 3 repetitions at a time.

[00355] Using the online pepsin / protease XIII column coupled with MSE data acquisition, a total of 109 peptides from hMUC16.nub were identified in the absence or presence of H4sH8767P, representing 64% sequence coverage. Six peptides had significantly reduced deuteration uptake (delta centroid values ​​> 0.5 daltons at least one point in the Petition 870260046565, dated 05 / 15 / 2026, page 167 / 435 159 / 202 time with p-values ​​< 0.05) when bound to H4sH8767P and are illustrated in Table 9A. The recorded peptide mass corresponds to the average mass value of MH+centroid from three replicates. These peptides, corresponding to amino acids 428 to 434, 453 to 467, and 474 to 481 of hMuc16.nub, had slower deuteration rates when bound to H4sH8767P. These identified residues also correspond to residues 14237 to 14243, 14262 to 14276, and 14283 to 14290 of hMUC16 as defined by Uniprot entry Q8WXI7 (MUC16_HUMAN), SEQ ID NO:1899. TABLE 9A. PEPTIDES OF hMUC16.nub WITH ALTERED DETERATION RATES UPON BINDING TO H4sH8767P Residues of SEQ ID NO: 1902 Deuteration of 1 min Deuteration of 20 min Amino acid sequence hMUC16 .nub hMUC16.nub + H4sH8767 P Δ hMUC16.nub hMUC16. nub + H4sH876 7P Δ 428 to 434 LYKGSQ L 809.97 ± 0.03 809.07± 0.06 - 0.26 811.05± 0.16 810.17± 0.01 - 0.88 429 to 434 YKGSQL 697.10 ± 0.00 696.74± 0.00 - 0.35 698.13± 0.02 697.60± 0.03 - 0.52 453-467 VTVKAL FSSNLD PSL 1595.35 ± 0.08 1593.97± 0.2 - 1.38 1596.01± 0.08 1595.33± 0.03 - 0.68 459-467 FSSNLD PSL 983.19 ± 0.01 981.57± 0.08 - 1.62 983.51±0 .03 982.76± 0.07 - 0.75 460-467 SSNLDP SL 835.44 ± 0.01 834.01± 0.00 - 1.43 835.89± 0.01 835.12± 0.15 - 0.74 474-481 DKTLNA SF 899.76 ± 0.00 899.25± 0.06 - 0.51 900.63 ± 0.00 900.10 ± 0.03 - 0.54

[00356] Using the online pepsin / protease XIII column coupled with MSE data acquisition, a total of 109 hMUC16.nub peptides were identified in the absence or presence of H1H8794P2, representing 64% sequence coverage. Three peptides showed significant deuteration uptake. Petition 870260046565, dated 05 / 15 / 2026, page 168 / 435 160 / 202 reduced (delta centroid values ​​> 0.5 daltons from at least one point in time with p-values ​​< 0.05) when linked to H1H8794P2 and are illustrated in Table 9B. The recorded peptide mass corresponds to the average mass value of the MH+ centroid of three replicates. These peptides, corresponding to amino acids 126 to 131, 127 to 131, and 132 to 138 of hMuc16.nub, had slower deuteration rates when linked to H1H8794P2. These identified residues also correspond to residues 13935 to 13940, 13936 to 13940, and 13941 to 13947 of hMUC16 as defined by Uniprot entry Q8WXI7 (MUC16_HUMAN), SEQ ID NO:1899. TABLE 9B. PEPTIDES OF hMUC16.nub WITH ALTERED DETERATION RATES UPON BINDING TO H1H8794P2 Residues of SEQ ID NO: 1902 Deuteration of 1 min Deuteration of 20 min Amino acid sequence hMUC16.n ub hMUC16.nu b + H4sH8767P Δ hMUC16.nub hMUC16.nu b + H4sH8767P Δ 126 to 131 LRYMAD 771.41 ± 0.01 770.60 ± 0.04 -0.81 771.91 ± 0.04 770.76 ± 0.02 -1.15 127-131 RYMAD 658.03 ± 0.02 657.32 ± 0.01 -0.71 657.89 ± 0.01 657.27 ±0.01 -0.6 132-138 MGQPGS L 692.55 ± 0.02 691.42 ± 0.15 -1.13 692.61± 0.01 691.58 ±0.02 -1.03

[00357] Using the online pepsin / protease XIII column coupled with MSE data acquisition, a total of 109 hMUC16.nub peptides were identified in the absence or presence of H1H8799P2, representing 64% sequence coverage. Four peptides showed significantly reduced deuteration uptake (delta centroid values ​​> 0.5 daltons at at least one time point with p-values ​​< 0.05) when bound to H1H8799P2 and are illustrated in Table 9C. The recorded peptide mass corresponds to the mean value of Petition 870260046565, dated 05 / 15 / 2026, p. 169 / 435 161 / 202 mass of MH+centroid of three replicates. These peptides, corresponding to amino acids 357 to 369, 358 to 366, 358 to 369 and 361 to 369 of hMuc16.nub, had slower deuteration rates when linked to H1H8799P2. These identified residues also correspond to residues 14165 to 14178, 14166 to 14176, 14166 to 14178 and 14170 to 14178 of hMUC16 as defined by Uniprot entry Q8WXI7 (MUC16_HUMAN), SEQ ID NO:1899. TABLE 9C. PEPTIDES OF hMUC16.nub WITH ALTERED DETERATION RATES UPON BINDING TO H1H8799P2 Residues of SEQ ID NO: 1902 Deuteration of 1 min Deuteration of 20 min Amino Acid Sequence hMUC16.nub hMUC16.nub + H1H8799P2 Δ hMUC16. nub hMUC16.nub + H1H8799P2 Δ 357 to 369 LSQLTHGV TQLGF 1404.15± 0.03 1403.41 ±0.09 -0.74 1406.14± 0.15 1404.26± 0.02 -2.11 358-366 SQLTHGVT QL 972.37± 0.10 972.04 ±0.10 -0.33 973.94± 0.03 972.56 ± 0.00 -1.38 358-369 SQLTHGVT QLGF 1291.23± 0.02 1290.20 ±0.00 -1.03 1291.34 ± 0.02 1291.05 ± 0.06 -2.27 361-369 THGVTQLG F 1404.15 ± 0.03 1403.42 ± 0.05 -0.73 1406.14 ± 0.14 1404.03 ± 0.02 -2.11 EXAMPLE 7: PHARMACOKINETIC EVALUATION OF ANTIBODIES BIESPECIFIC ANTI-MUC16 x CD3

[00358] The pharmacokinetic evaluation of bispecific anti-MUC16 x CD3 antibodies BSMUC16 / CD3-001 and BSMUC16 / CD3-005 and an isotype control were conducted in humanized MUC16 x CD3 mice (mice homozygous for human MUC16 and CD3 expression, MUC16hu / hu x CD3hu / hu), humanized CD3 mice (mice homozygous for human CD3 expression, CD3hu / hu), and wild-type (WT) strain mice. Petition 870260046565, dated 05 / 15 / 2026, p. 170 / 435 162 / 202 corresponding (75% C57BL, 25% 129Sv). Cohorts contained 4 to 5 mice per antibody tested and per mouse strain. All mice received a single intraperitoneal (ip) dose of 0.4 mg / kg. Blood samples were collected at 3 and 6 hours, 1, 3, 7, 14, and 28 days after dosing. Blood was processed into serum and frozen at -80 °C until analysis.

[00359] Circulating antibody concentrations were determined by total human IgG antibody analysis using GyroLab xPlore™ (Gyros, Uppsala, Sweden). Briefly, a biotinylated goat anti-human IgG polyclonal antibody (Jackson ImmunoResearch, West Grove, PA) was captured on streptavidin-coated microspheres in a Gyrolab Bioaffy 200 CD (Gyros) to capture human IgG present in sera. After affinity column capture, bound human IgG antibody in samples was detected with Alexa-647 identified goat anti-human IgG (Jackson ImmunoResearch). The fluorescent signal in the column allowed the detection of bound IgG, and response units (RU) were read by the instrument. Sample concentrations were determined by interpolation of a standard curve that was fitted using a 5-parameter logistic curve fitting with the Gyrolab Evaluator software.PK parameters were determined by non-compartmental analysis (NCA) using Phoenix®WinNonlin® Version 6.3 software (Certara, LP, Princeton, NJ) and an extravascular dosing model. Using the respective mean concentration values ​​for each antibody, all pharmacokinetic parameters, including maximum observed serum concentration (Cmax), estimated observed half-life (t1 / 2), and area under the concentration versus time curve to last measurable concentration (AUClast), were determined using a linear trapezoidal rule with linear interpolation and uniform weighting. Petition 870260046565, dated 05 / 15 / 2026, page 171 / 435 163 / 202

[00360] Following ip administration of antibodies in WT mice, the concentration-time profiles of total IgG of BSMUC16 / CD3001, BSMUC16 / CD3-005 and the isotype control were all similar, characterized first by a brief drug distribution followed by a single drug elimination phase throughout the remainder of the study. The maximum serum concentrations (Cmax) and calculated drug exposure (AUClast) of the three antibodies were comparable (in multiplications of 1.3 of each other).

[00361] Following ip administration of antibodies in CD3hu / hu mice, BSMUC16 / CD3-001, BSMUC16 / CD3-005 and the isotype control had comparable Cmax concentrations (4.6, 3.6 and 4.1 μg / mL, respectively). BSMUC16 / CD3-005 and the isotype control exhibited similar drug clearance curves, while BSMUC16 / CD3001 exhibited steeper drug clearance than both, suggesting that binding to the human CD3 target triggers clearance. The terminal antibody concentration for BSMUC16 / CD3-001 was 0.03 μg / mL, which is approximately 28 times lower than the terminal antibody concentrations determined for the isotype control (0.85 μg / mL) and 22 times lower than serum concentrations of BSMUC16 / CD3-005 (0.66 pg / mL).

[00362] In MUC16hu / hux CD3hu / hu doubly humanized mice, Muc16xCD3 bispecific and isotope control antibodies had comparable Cmax concentrations (Cmax range: 4.5 to 6.9 pg / mL). Both bispecific antibodies exhibited steeper drug clearance than the isotype control, suggesting a target-mediated effect. Terminal antibody concentrations for BSMUC16 / CD3-001 and BSMUC16 / CD3-005 were approximately 29 times and 2.9 times lower, respectively, than the terminal antibody concentrations determined for the isotype control (0.86 pg / mL). Petition 870260046565, dated 05 / 15 / 2026, p. 172 / 435 164 / 202

[00363] A summary of the data for total concentrations of bispecific anti-MUC16 x CD3 antibodies and isotype control antibody is summarized in Table 10. Mean PK parameters are described in Tables 11A and 11B. Mean total antibody concentrations versus time are shown in Figures 1, 2, and 3. In conclusion, the bispecific MUC16 x CD3 antibodies exhibited similar Cmax and drug elimination curves in WT mice; however, BSMUC16 / CD3-001 exhibited steeper elimination rates than BSMUC16 / CD3-005, and the isotype control consisted of CD3-only humanized mice and MUC16 / CD3-doubly humanized mice. Since the bispecific antibodies administered in this PK study comprise the same anti-MUC16 binding arm, the results suggest that the binding strength of the CD3 targeting arm may play a role in drug exposure levels (AUClast) and drug elimination rates.Neither BSMUC16 / CD3-001 nor BSMUC16 / CD3005 binds to mouse MUC16 or mouse CD3. TABLE 10: MEAN CONCENTRATIONS OF TOTAL IgG IN SERUM AFTER A SINGLE INTRAPERITONEAL INJECTION OF 0.4 mg / kg OF BSMUC16 / CD3-001, BSMUC16 / CD3-005 AND ANTIBODIES OF ISOTOPE CONTROL IN WT MICE, HUMANIZED CD3 MICE AND HUMANIZED MUC16 x CD3 MICE Antibody Time (d) Total mAb concentration in mouse serum WT CD3hu / hu MUC16hu / hu x CD3hu / hu Average (pg / mL) + / - SD Average (pg / mL) + / - SD Average (pg / mL) + / - SD BSMUC16 / C 0.13 5.39 0.34 4.30 0.29 6.77 1.52 Petition 870260046565, dated 05 / 15 / 2026, p. 173 / 435 165 / 202 Antibody D3-001 Time (d) Total mAb concentration in mouse serum WT CD3hu / hu MUC16hu / hu x CD3hu / hu Average (pg / mL) + / - SD Average (pg / mL) + / - SD Average (pg / mL) + / - SD 0.25 5.80 0.36 4.26 1.07 6.63 1.06 1.00 4.13 0.43 2.87 0.71 4.89 0.53 3.00 3.19 0.53 1.44 0.27 2.50 0.22 7.00 2.61 0.73 0.72 0.13 1.20 0.22 14.00 1.44 0.69 0.18 0.05 0.28 0.08 21.00 0.93 ND 0.07 0.02 0.06 0.05 28.00 0.60 ND 0.04 0.01 0.03 0.02 BSMUC16 / C D3-005 0.13 4.23 0.62 3.35 1.15 4.35 0.24 0.25 4.53 0.55 3.40 0.96 4.45 0.49 1.00 3.47 0.32 2.72 0.42 3.00 0.61 3.00 2.51 0.13 1.95 0.37 1.98 0.41 7.00 2.02 0.24 2.31 0.67 1.58 0.36 14.00 1.19 0.17 1.01 0.23 0.78 0.26 21.00 1.19 0.29 1.19 0.11 0.66 0.29 28.00 0.71 0.20 0.66 0.28 0.30 0.22 Isotype Control 0.13 5.07 1.16 5.43 1.30 6.56 0.70 0.25 5.91 1.10 5.67 1.91 6.48 0.90 1.00 2.64 0.24 2.98 1.14 2.82 0.30 Petition 870260046565, dated 05 / 15 / 2026, p. 174 / 435 166 / 202 Antibody Time (d) Total mAb concentration in mouse serum WT CD3hu / hu MUC16hu / hu x CD3hu / hu Average (pg / mL) + / - SD Average (pg / mL) + / - SD Average (pg / mL) + / - SD 3.00 2.05 0.06 2.29 0.83 1.57 0.37 7.00 1.80 0.25 2.14 0.85 1.96 0.37 14.00 1.22 0.28 1.48 0.66 1.34 0.37 21.00 1.20 0.58 1.43 0.72 1.24 0.44 28.00 0.73 0.24 0.85 0.29 0.86 0.41 Time: (h, when observed) = time in hours after single dose injection; D = Study day; SD = Standard deviation; ND = Not determined due to exclusion of mice with drug clearance antidrug titers TABLE 11A: SUMMARY OF PHARMACOKINETIC PARAMETERS: Humanized mice from CD3hu / hu Parameter Unit s Mice WT CD3hu / hu Mice Isotype Control BSMUC16 / CD3-001 BSMUC16 / CD3-005 Isotype Control BSMUC16 / CD3-001 BSMUC16 / CD3-005 Cmax pg / mL 5 ± 3 6 ± 0.4 5 ± 0.5 4.1 ± 3 4.6 ± 0.8 3.5 ± 1 T1 / 2 d 11 ± 4 7 ± 3 12 ± 2 14 ± 0.5 3.9 ± 0.6 11 ± 5 AUClast d^pg / mL 35 ± 18 40 ± 11 45 ± 5 49 ± 20 16 ± 3 36 ± 13 Cmax = Peak concentration; AUC = Area under the concentration-time curve; AUClast = AUC calculated from time zero to the time of the last positive concentration; T1 / 2 = Estimated observed half-life TABLE 11B: SUMMARY OF PHARMACOKINETIC PARAMETERS: DOUBLE-HUMANIZED MICE FROM MUC16hu / hux CD3hu / hu Petition 870260046565, dated 05 / 15 / 2026, p. 175 / 435 167 / 202 Parameter Unit s Mice WT Mice with MUC16 hu / hu x CD3 hu / hu Isotype Control BSMUC16 / CD3-001 BSMUC16 / CD3-005 Isotype Control BSMUC16 / CD3-001 BSMUC16 / CD3-005 Cmax μg / mL 5 ± 3 6 ± 0.4 5 ± 0.5 6.7 ± 0.7 6.9 ± 1 4.5 ± 4 T1 / 2 d 11 ± 4 7 ± 3 12 ± 2 12.9 ± 4 3.3 ± 0.8 8.2 ± 4 AUClast d^g / mL 35 ± 18 40 ± 11 45 ± 5 46 ± 10 27 ± 3 34 ± 11 Cmax = Peak concentration; AUC = Area under the concentration-time curve; AUClast = AUC calculated from time zero to the time of the last positive concentration; T1 / 2 = Estimated observed half-life EXAMPLE 8: Bispecific anti-MUC16 / anti-CD3s antibodies exhibit potent anti-tumor efficacy in vivo.

[00364] To determine the in vivo efficacy of exemplary bispecific anti-MUC16 / anti-CD3 antibodies identified as having weak or undetectable binding to human CD3 or cynomolgus, studies were conducted in immunocompromised mice bearing human prostate cancer xenografts. The efficacy of selected bispecific antibodies was tested in both immediate treatment and therapeutic dosing models. EFFICACY OF BIESPECIFIC ANTI-MUC16 / ANTICD3 ANTIBODIES IN HUMAN TUMOR XENOGRAFT MODELS

[00365] To evaluate the in vivo efficacy of bispecific anti-MUC16 / anti-CD3 antibodies in human tumor xenograft studies, NOD scid gamma (NSG) mice (Jackson Laboratories, Bar Harbor, Maine) were pre-implanted with human peripheral blood mononuclear cells (PBMCs; ReachBio LLC., Seattle, WA) and then received luciferase-transduced human ovarian cancer cell line (OVCAR-3 / Luc) ascites cells from the OVCAR-3 human ovarian cancer cell line (American Type Tissue Culture, Manassas, VA). OVCAR-3 type cells endogenously express MUC-16. Petition 870260046565, dated 05 / 15 / 2026, page 176 / 435 168 / 202

[00366] Briefly, NSG mice were injected intraperitoneally (ip) with 5.0 x 106 human PBMCs. 8 days later, 1.5 x 106 ascites cells of the OVCAR3 / Luc cell line, previously passed in vivo, were administered ip to PBMC-engraved NSG mice. In the immediate treatment group, mice were treated ip on the day of OVCAR-3 / Luc cell implantation with bispecific MUC16 / CD3 antibodies BSMUC16 / CD3-001 or BSMUC16 / CD3-005, or an isotype control, at a dose of 10 pg / mouse (N=5 mice / treatment group). In the therapeutic dose model, mice were treated ip 7 days after tumor implantation with the bispecific MUC16 / CD3 antibodies or control antibodies described above, at a dose of 10 pg / mouse (N=5 / treatment group).

[00367] In all studies, tumor growth was monitored using bioluminescent imaging (BLI). Mice were injected intraperitoneally (ip) with the luciferase substrate D-luciferin suspended in PBS (150 mg / kg) and imaged under isoflurane anesthesia after 10 min. BLI was performed using the Xenogen IVIS system (Perkin Elmer, Hopkinton, MA), and BLI signals were extracted using Living Image software (Xenogen / Perkin Elmer). Regions of interest were drawn around each cell mass, and photon intensities were recorded as photons (p) / s (s) / cm2 / steradian (sr). For the immediate treatment group, data are shown as BLI levels 26 days after tumor implantation (Table 12A). For the therapeutic treatment group, data are shown as the change in BLI multiplication between day 6 (1 day before treatment) and the study endpoint (26 days after tumor implantation; Table 12B).

[00368] As the results show, both BSMUC16 / CD3-001 and BSMUC16 / CD3-005 showed similar efficacy in suppressing tumor growth compared to the isotype control when Petition 870260046565, dated 05 / 15 / 2026, page 177 / 435 169 / 202 BLI was measured on Day 26 in the immediate dosing model. Both bispecific anti-MUC16 / anti-CD3 antibodies also suppressed the growth of established tumors when administered 7 days after tumor implantation, compared to the control. In summary, the bispecific anti-MUC16 / anti-CD3 antibody of this invention exhibits potent anti-tumor efficacy in various models. TABLE 12A: EFFICACY OF BI-SPECIFIC ANTI-MUC16 / ANTI-CD3 ANTIBODIES IN A XENOGRAFT MODEL IMMUNOCOMPROMISED: IMMEDIATE DOSAGE Tumor Model / Mouse Strain / Dose Bispecific Antibody Identifier N Average Bioluminescent Radiance (photons / s / cm2 / steraday on) Day 26 (mean ± SD) OVCAR-3 / Luc / NSG / 10 pg / mouse BSMUC16 / CD3-001 5 1.4 x 103 ± 3.5 x 102 BSMUC16 / CD3-005 5 1.5 x 103 ± 9.7 x 102 Isotype Control 5 2.0x107 ± 1.0x106 TABLE 12B: EFFICACY OF BIESPECIFIC ANTIBODY ANTIBODIES MUC16 / ANTI-CD3 IN A XENOGRAFT MODEL IMMUNOCOMPROMISED: THERAPEUTIC TREATMENT Tumor Model / Mouse Strain / Dose Bispecific Antibody Identifier N Change in multiplication in mean Bioluminescent Radiance [p / s / cm2 / sr] on Day 26 compared to Day 6 (mean ± SD) OVCAR-3 / Luc / BSMUC16 / CD3-001 5 2.0 ± 5.0 Petition 870260046565, dated 05 / 15 / 2026, p. 178 / 435 170 / 202 Tumor Model / Mouse Strain / Dose Bispecific Antibody Identifier N Change in multiplication in Bioluminescent Radiance of mean [p / s / cm2 / sr] on Day 26 compared to Day 6 (mean ± SD) NSG / 10 pg / mouse BSMUC16 / CD3-005 5 0.01 ± 0.02 Isotype Control 5 21.0 ± 8.0

[00369] In further experiments, the in vivo efficacy of a bispecific anti-MUC16 / anti-CD3 antibody was evaluated in xenogeneic and syngeneic tumor models. For the first xenogeneic model, NSG mice were intraperitoneally (IP) injected with previously passed OVCAR-3 / Luc cells in vivo (Day 0) eleven days after grafting with human PBMCs. Mice were treated IP with 0.01, 0.1, or 0.5 mg / kg BSMUC16 / CD3-001, or administered 0.5 mg / kg of non-binding control or CD3-binding control on Days 6, 10, 13, 16, and 21. Tumor burden was assessed by BLI on Days 6, 14, and 20 after tumor implantation. Treatment with 0.1 or 0.5 mg / kg of BSMUC16 / CD3-001 resulted in significant antitumor efficacy as determined by BLI measurements on Day 20, as shown in Tables 13A to 13C and Figures 4 to 6.For the second xenogeneic model, NSG mice were injected with previously passed OVCAR-3 / Luc cells in vivo (Day 0) thirteen days after grafting with human PBMCs, and a second batch of PBMCs was transferred on Day 4. Mice were treated intravenously (IV) with 0.1, 0.5, 1, or 5 mg / kg BSMUC16 / CD3-005 or administered 5 mg / kg of a non-binding control or a CD3-binding control on Days 5, 8, 12, 15, 19, and 22. Tumor burden was assessed by BLI on Days 4, 11, 18, and 25. Petition 870260046565, dated 05 / 15 / 2026, page 179 / 435 171 / 202 with 0.5, 1, or 5 mg / kg of BSMUC16 / CD3-005 resulted in significant antitumor efficacy as shown by BLI measurements and multiplication changes (Tables 13D to 13F and Figures 7 to 9). To examine efficacy in an immunocompetent model, the murine CD3 gene was replaced with human CD3, and a portion of the mouse MUC16 gene was replaced with the human sequence. The substitutions resulted in a mouse whose T cells express human CD3 and which expresses a chimeric MUC16 molecule containing a portion of human MUC16 to which the bispecific antibody BSMUC16 / CD3-001 binds. For the syngeneic tumor model, ID8-VEGF cell lines modified to express the human MUC16 portion were used. Mice were implanted with ID8-VEGF / huMUC16 cells subcutaneously and treated with 100 μg of BSMUC16 / CD3-001 on the day of implantation or ten days after implantation, when tumors were established.Treatment with 100 μg of BSMUC16 / CD3-001 resulted in significant anti-tumor efficacy, as shown in Table 13G and Figure 10.

[00370] Xenograft tumor implantation and measurement: Ascites cells of the OVCAR-3 / Luc cell line, previously passed in vivo, were administered IP into NSG mice previously grafted with human PBMCs. BLI was measured as a readout for tumor growth several days after OVCAR-3 / Luc implantation and at multiple times during the study. After initial cohort BLI measurement, mice were divided into groups of 4 to 6 animals each and administered bispecific MUC16xCD3 or control antibodies twice weekly throughout the study.

[00371] Calculation of xenograft tumor growth and inhibition: bioluminescence imaging was used to measure tumor burden. Mice were injected with IP at 150 mg / kg (as determined Petition 870260046565, dated 05 / 15 / 2026, p. 180 / 435 172 / 202 by body weights at the start of the experiment) of the luciferase substrate D-luciferin suspended in PBS. Ten minutes after dosing, BLI imaging of the mice was performed under isoflurane anesthesia using the Xenogen IVIS system. Image acquisition was performed with the field of view in D, subject height of 1.5 cm, and half linkage level for an exposure time of 0.5 min. BLI signals were extracted using Living Image software. Regions of interest were drawn around each tumor mass and photon intensities were recorded as p / s / cm² / sr. Statistical analysis was performed using GraphPad Prism software (Version 6). Statistical significance for the BLI results was determined using an unpaired, non-parametric Mann-Whitney t-test. The multiplication changes were calculated using the formula: (Day20-Day6) / Day6 for study 1 and (Day25-Day4) / Day4 for study 2.

[00372] Implantation and measurement of syngeneic tumors: Mice expressing human CD3 and a human-murine chimera of MUC16 at the corresponding mouse loci were implanted with 10e6 ID8-VEGF / huMUC16 cells subcutaneously (SC). Mice were administered BSMUC16 / CD3-001 or a CD3-binding IP control twice weekly throughout the study. Treatment began on Day 0 or Day 10 after implantation. Tumor growth was measured with calipers twice weekly. Mice were sacrificed 47 days after tumor implantation.

[00373] Calculation of syngeneic tumor growth and inhibition: In order to determine the tumor volume with an external caliper, the largest longitudinal diameter (length) and the largest transverse diameter (width) were determined. The tumor volume based on caliper measurements was calculated using the formula: Volume = Petition 870260046565, dated 05 / 15 / 2026, page 181 / 435 173 / 202 (length x width2) / 2. Statistical significance was determined using a non-parametric unpaired Mann-Whitney t-test.

[00374] The anti-tumor efficacy of the bispecific antibody BSMUC16 / CD3-001 in xenogeneic and syngeneic tumor models in vivo is shown in Tables 13A to D below. TABLE 13A: OVCAR-3 MODEL STUDY 1. BIOLUMINESCENCE LEVEL ON DAY 6 AFTER TUMOR IMPLANTATION Antibody (mg / kg) Mean Radiance [p / s / cm22 / sr] 6 days post-implantation (mean ± SEM) Non-binding control (0.5) 8.15e+05 ± 7.88e+04 CD3 binding control (0.5) 6.39e+05 ± 8.67e+04 BSMUC16 / CD3-001 (0.5) 7.64e+05 ± 1.19e+05 BSMUC16 / CD3-001 (0.1) 6.31e+05 ± 1.10e+05 BSMUC16 / CD3-001 (0.01) 8.77e+05 ± 7.91e+04 TABLE 13B: OVCAR-3 MODEL STUDY 1. LEVEL OF Bioluminescence on day 20 after tumor implantation. Antibody (mg / kg) Mean Radiance [p / s / cm22 / sr] 20 days post-implantation (mean ± SEM) Non-binding control (0.5) 8.63e+06 ± 1.45e+06 CD3 binding control (0.5) 9.94e+06 ± 1.08e+06 BSMUC16 / CD3-001 (0.5) 9.37e+02 ± 9.62e+02 BSMUC16 / CD3-001 (0.1) 2.36e+04 ± 1.28e+06 BSMUC16 / CD3-001 (0.01) 6.51e+06 ± 1.60e+06 TABLE 13C: OVCAR-3 MODEL STUDY 1. CHANGE IN BLI MULTIPLICATION BETWEEN DAY 6 AND DAY 20 AFTER THE Petition 870260046565, dated 05 / 15 / 2026, page 182 / 435 174 / 202 TUMOR IMPLANTATION Antibody (mg / kg) Change in multiplication in Mean Radiance [p / s / cm22 / sr] from Day 6 to Day 20 post-implantation (mean ± SD) Non-binding control (0.5) 9.5 ± 1.9 CD3 binding control (0.5) 15.6 ± 6.7 BSMUC16 / CD3-001 (0.5) -1.00 ± 0.00 BSMUC16 / CD3-001 (0.1) 1.2 ± 4.7 BSMUC16 / CD3-001 (0.01) 5.6 ± 4.2 TABLE 13D: OVCAR-3 MODEL STUDY 2. LEVEL OF Bioluminescence on day 4 after tumor implantation. Antibody (mg / kg) Mean Radiance [p / s / cm22 / sr] 4 days post-implantation (mean ± SEM) Non-binding control (5) 1.54e+05 ± 9.93e+03 CD3 binding control (5) 1.34e+05 ± 1.55e+04 BSMUC16 / CD3-005 (5) 1.54e+05 ± 1.03e+04 BSMUC16 / CD3-005 (1) 1.38e+05 ± 4.65e+03 BSMUC16 / CD3-005 (0.5) 1.31e+05 ± 4.03e+03 BSMUC16 / CD3-005 (0.1) 1.53e+05 ± 1.93e+04 TABLE 13E: OVCAR-3 MODEL STUDY 2. LEVEL OF Bioluminescence on day 25 after tumor implantation. Petition 870260046565, dated 05 / 15 / 2026, page 183 / 435 175 / 202 Antibody (mg / kg) Mean Radiance [p / s / cm22 / sr] 25 days post-implantation (mean ± SEM) Non-binding control (5) 7.20e+06 ± 8.91e+05 CD3 binding control (5) 6.15e+06 ± 7.26e+05 BSMUC16 / CD3-005 (5) 1.52e+03 ± 4.86e+05 BSMUC16 / CD3-005 (1) 6.99e+03 ± 6.23e+03 BSMUC16 / CD3-005 (0.5) 2.23e+03 ± 2.35e+05 BSMUC16 / CD3-005 (0.1) 7.63e+06 ± 1.83e+06 TABLE 13F: OVCAR-3 MODEL STUDY 2. CHANGE IN BLI MULTIPLICATION BETWEEN DAY 4 AND DAY 25 AFTER TUMOR IMPLANTATION Antibody (mg / kg) Change in multiplication in Mean Radiance [p / s / cm22 / sr] from Day 4 to D25 post-implantation (mean ± SD) Non-binding control (5) 46.8 ± 20.6 CD3 binding control (5) 55.0 ± 14.7 BSMUC16 / CD3-005 (5) 2.5 ± 8.5 BSMUC16 / CD3-005 (1) -0.9 ± 0.1 BSMUC16 / CD3-005 (0.5) 0.7 ± 3.6 BSMUC16 / CD3-005 (0.1) 45.4 ± 35.7 TABLE 13G: ID8-VEGF / huMUC16 MODEL. SIZE OF Tumor (mm3) on day 47 Petition 870260046565, dated 05 / 15 / 2026, page 184 / 435 176 / 202 Treatment Initiation Antibody ^g) Tumor Size (mm3) on Day 47 (mean ± SEM) Day 0 CD3 Binding Control (100) 827.5 ± 223.5 Day 0 BSMUC16 / CD3-001 (100) 51.2 ± 51.2 Day 10 BSMUC16 / CD3-001 (100) 273.8 ± 92.36 EXAMPLE 9: CONJUGATE PREPARATION AND CHARACTERIZATION

[00375] All monoclonal antibodies were expressed in CHO cells and purified by Protein A. An isotype control was also prepared in a similar manner. The non-binding isotype control antibody was derived from an immunological antigen unrelated to oncology.

[00376] The antibody (10 mg / mL) in 50 mM HEPES, 150 mM NaCl, pH 7.5, was treated with 1 mM dithiothreitol at 37 °C for 30 min. After gel filtration (G-25, pH 4.5 sodium acetate), one of the maleimide ligand load derivatives Compound 7 or Compound 10 (see Table 14) (1.2 equivalents / SH group) in DMSO (10 mg / mL) was added to the reduced antibody and the mixture adjusted to pH 7.0 with 1 M HEPES (pH 7.4). Compound 7 and Compound 10, and the methods for producing the compounds, are described in PCT Publication WO2014 / 145090, published on September 18, 2014, and PCT Publication WO2016 / 160615, published on October 6, 2016, respectively, each of which is incorporated herein by reference. After 1 h, the reaction was rapidly cooled with excess N-ethyl maleimide. The conjugates were purified by size exclusion chromatography and filtered to sterility.Protein and ligand loading concentrations were determined by UV spectral analysis. Size-exclusion HPLC established that all conjugates... Petition 870260046565, dated 05 / 15 / 2026, page 185 / 435 Of the 177 / 202 antibodies used, >95% were monomeric. Yields are reported in Table 14 based on protein determinations. All conjugated antibodies were analyzed by UV for ligand loading values ​​according to Hamblett et al., Cancer Res 2004 10 7063. Results are summarized in Table 14.

[00377] A conjugate comprising Compound 60 can be prepared using a similar method. Compound 60 and methods for producing the compound are described in PCT Publication No. W02016 / 160615 (Example 20), published on October 6, 2016, which is incorporated herein by reference in its entirety. Compound 60 is Maitansin-N-methyl-L-alanine-(3-methoxy-4-amino)benzamido-Cit-Val-Cap-Mal. TABLE 14: SUMMARY OF LOAD (CHEMOTOXIC DRUG) AND ANTIBODY-DRUG CONJUGATE PARAMETERS Compound ε252 nm (cm-1 M'1) ε280 nm (cm-1 M'1) 7 [Maitansin-3-N-methyl-L-(S)-alaninepropanamidyl-3-N-methyl-N-[4-(aminocitrulline-valine-hexanamide-6maleimidyl)benzyl]carbamate] 50600 8100 10 [Maitansin-N-methyl-L-alanine-4aminobenzamide-citrulline-valinacaprolyl-6-maleimidyl] 45990 20600 Antibody ε252 nm (cm-1 M'1) ε280 nm (cm-1 M'1) H1H9519N 83995 235280 H1H9521N 85564 232050 Isotype Control 75113 218360 Antibody Conjugate Load: Antibody (UV) % Yield H1H9519N-7 3.5 40 Petition 870260046565, dated 05 / 15 / 2026, page 186 / 435 178 / 202 Compound ε252 nm (cm-1 M'1) ε280 nm (cm-1 M'1) H1H9521N-7 3.6 40 H1H9521N-10 3.0 40 Isotype-7 control 3.0 60 Isotype-10 control 3.4 60 EXAMPLE 10: ANTI-ANTIBODY DRUG CONJUGATES MUC16 are potent inhibitors of tumor growth in MUC16-positive prostate cancer xenograft models in vivo.

[00378] To determine the in vivo efficacy of anti-MUC16 antibodies conjugated to Compound 7 and Compound 10, studies were conducted in immunocompromised mice bearing MUC16-positive ovarian cancer xenografts.

[00379] For these studies, female SCID mice (Taconic, Hudson NY) were implanted with luciferase-transfected cells (OVCAR3 / luc) containing OVCAR3 [NIHOVCAR-3 (OVCAR3, ATCC HTB161)] that endogenously express MUC16. For intraperitoneal (IP) tumors, mice were randomized into treatment groups and dosed with anti-MUC16 drug antibody conjugates (see Example 9), a non-binding antibody conjugate, or vehicle after detection of luminescent tumor signal. For subcutaneous (SC) xenografts, once tumors reached a mean volume of 200 mm3 (~Day 16), mice were randomized into treatment groups and dosed with anti-MUC16 drug antibody conjugates, a non-binding antibody conjugate, or vehicle. In these in vivo studies, antibodies were measured and the tumors were then monitored until ascites developed or an average tumor size of approximately 1.200 mm3 was achieved in the cohort dosed with vehicle alone. At that point, Tumor Growth Inhibition was achieved. Petition 870260046565, dated 05 / 15 / 2026, page 187 / 435 179 / 202 calculated.

[00380] In an initial intraperitoneal (IP) study, exemplary anti-MUC16 antibodies conjugated to Compound 7 were examined for efficacy in reducing OVCAR3 / luc luminescence signal. Mice received four once-weekly doses of anti-MUC16 and control ADCs at 85 μg / kg drug equivalent based on ADC:antibody drug ratios. As summarized in Table 15A, H1H9519N-Compound 7 and H1H9521N-Compound 7 potentially inhibited ascites tumor growth. These anti-MUC16 ADCs effectively reduced tumor size, with a 100 percent reduction in tumor luminescence compared to the vehicle control. The Control ADC did not mediate any inhibition of OVCAR / luc ascites tumor cell growth.

[00381] An additional study evaluating the efficacy of anti-MUC16 ADCs against subcutaneous (SC) OVCAR3 / luc tumor is summarized in Table 15B. Mice received four once-weekly doses of anti-MUC16 and control ADCs at 85 μg / kg drug equivalent based on ADC:antibody drug ratios. When conjugated to Compound 7, MUC16 ADCs, H1H9519N-Compound 7 and H1H9521N-Compound 7 again produced a significant antitumor effect; this time, against tumors of Subcutaneous OVCAR3 / luc. Consequently, these ADCs mediated a 100% and 109% inhibition of tumor growth, respectively. The Control ADC did not mediate any inhibition of subcutaneous OVCAR / luc tumor growth.

[00382] In a third study, the efficacy of the anti-MUC16 antibody H1H9521N conjugate to the Compound 10 ligand drug was evaluated in the OVCAR3 / luc IP tumor model. Mice received single doses of anti-MUC16 and control ADCs at 85 Petition 870260046565, dated 05 / 15 / 2026, page 188 / 435 180 / 202 μg / kg, 170 μg / kg, and 340 μg / kg of drug equivalent based on ADC:antibody drug ratios. As summarized in Table 15C, H1H9519N-10 potently inhibited ascites tumor growth. Doses of H1H9519N-Compound 10 resulted in 99 to 100% inhibition of tumor luminescence relative to the vehicle control. Some inhibition was observed with Control ADC using Compound 10, although this was more moderate than that observed after H1H9519N anti-MUC16-Compound 10. TABLE 15A: INHIBITION OF OVCAR3 / luc IP TUMOR GROWTH ON DAY 49 IN SCID MICE TREATED WITH ANTI-MUC16 ANTIBODIES CONJUGATED TO COMPOUND 7 Treatment Group Mean Radiance of Final Tumor (mean ± SEM) Mean Tumor Growth Inhibition (%) Vehicle 16469750±10679335 0 Control-Compound 7 85 μg / kg 16813750 ±4026065 - 2 H1H9519N-Compound 7 85 μg / kg 111254±187288 100 H1H9521N-Compound 7 85 μg / kg 110413 ±161353 100 TABLE 15B: INHIBITION OF OVCAR3 / luc SC TUMOR GROWTH ON DAY 37 IN SCID MICE TREATED WITH ANTI-MUC16 ANTIBODIES CONJUGATED TO COMPOUND 7 Treatment Group | Final Tumor Volume (mean ± SEM) | Mean Tumor Growth Inhibition (%) | Vehicle | 1210 ±426 | 0 | Control-Compound | 7 | 85 μg / kg | 1737 ±391 | -51 Petition 870260046565, dated 05 / 15 / 2026, page 189 / 435 181 / 202 H1H9519N- Compound 7 85 μg / kg 187±269 100 H1H9521N- Compound 7 85 μg / kg 89 ± 97 109 TABLE 15C: INHIBITION OF OVCAR3 / luc IP TUMOR GROWTH ON DAY 49 IN SCID MICE TREATED WITH ANTI-MUC16 ANTIBODIES CONJUGATED TO COMPOUND 10 Treatment Group Final Tumor Radiance (mean ± SEM) Mean Tumor Growth Inhibition (%) Vehicle 29211000±23504780 0 Control-Composite 10 85 μg / kg 17332625 ±14346694 41 Control-Composite 10 170 μg / kg 32075000±15623403 -10 Control-Composite 10 340 μg / kg 22882350±18771913 22 H1H9521N-Composite 10 85 μg / kg 574285 ± 306844 99 H1H9521N-Composite 10 170 μg / kg 236037 ± 226948 100 H1H9521N- Compound 10 340 μg / kg 26472 ± 25079 101 EXAMPLE 11: GENERATION OF ANTI-CD3 ANTIBODIES

[00383] Anti-CD3 antibodies were obtained by immunizing a modified mouse containing DNA encoding variable regions of human immunoglobulin kappa heavy and light chains with cells expressing CD3 or with DNA encoding CD3. The antibody immune response was monitored by an immunoassay. Petition 870260046565, dated 05 / 15 / 2026, page 190 / 435 182 / 202 specific to CD3. When a desired immune response was achieved, splenocytes were collected and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. The hybridoma cell lines were examined and screened to identify cell lines that produce CD3-specific antibodies. Using this technique, several chimeric anti-CD3 antibodies (i.e., antibodies that have human variable domains and mouse constant domains) were obtained. In addition, several fully human anti-CD3 antibodies were isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in US patent 2007 / 0280945A1.

[00384] Certain biological properties of the exemplary anti-CD3 antibodies generated according to the methods in this Example are described in detail in the Examples in this document. EXAMPLE 12: VARIABLE REGION OF HEAVY AND LIGHT AMINO ACID CHAINS AND NUCLEIC ACID SEQUENCES

[00385] Table 16 presents amino acid sequence identifiers of variable heavy and light chain regions and CDRs of selected anti-CD3 antibodies from the invention. The corresponding nucleic acid sequence identifiers are presented in Table 17. The methods for producing the anti-CD3 antibodies disclosed herein can also be found in publication no. US 2014 / 0088295. TABLE 16: AMINO ACID SEQUENCE IDENTIFIERS SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H1H2712N 402 404 406 408 410 412 414 416 H1M2692N 418 420 422 424 426 428 430 432 Petition 870260046565, dated 05 / 15 / 2026, p. 191 / 435 183 / 202 SEQ ID NOs: Antibody Design HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H1M3542N 434 436 438 440 442 444 446 448 H1M3544N 450 452 454 456 458 460 462 464 H1M3549N 466 468 470 472 474 476 478 480 H1M3613N 482 484 486 488 490 492 494 496 H2M2689N 498 500 502 504 506 508 510 512 H2M2690N 514 516 518 520 522 524 526 528 H2M2691N 530 532 534 536 538 540 542 544 H2M2704N 546 548 550 552 554 556 558 560 H2M2705N 562 564 566 568 570 572 574 576 H2M2706N 578 580 582 584 586 588 590 592 H2M2707N 594 596 598 600 602 604 606 608 H2M2708N 610 612 614 616 618 620 622 624 H2M2709N 626 628 630 632 634 636 638 640 H2M2710N 642 644 646 648 650 652 654 656 H2M2711N 658 660 662 664 666 668 670 672 H2M2774N 674 676 678 680 682 684 686 688 H2M2775N 690 692 694 696 698 700 702 704 H2M2776N 706 708 710 712 714 716 718 720 H2M2777N 722 724 726 728 730 732 734 736 H2M2778N 738 740 742 744 746 748 750 752 H2M2779N 754 756 758 760 762 764 766 768 H2M2789N 770 772 774 776 778 780 782 784 H2M2862N 786 788 790792 794 796 798 800 H2M2885N 802 804 806 808 810 812 814 816 H2M2886N 818 820 822 824 826 828 830 832 Petition 870260046565, dated 05 / 15 / 2026, page 192 / 435 184 / 202 SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H2M3540N 834 836 838 840 842 844 846 848 H2M3541N 850 852 854 856 858 860 862 864 H2M3543N 866 868 870 872 874 876 878 880 H2M3547N 882 884 886 888 890 892 894 896 H2M3548N 898 900 902 904 906 908 910 912 H2M3563N 914 916 918 920 922 924 926 928 H1H5751P 930 932 934 936 938 940 942 944 H1H5752P 946 948 950 952 954 956 958 960 H1H5753B 962 964 966 968 970 972 974 976 1006 1008 H1H5756B 1010 1012 1014 1016 1018 1020 1022 1024 H1H5757B 1026 1028 1030 1032 1034 1036 1038 1040 H1H5758B 1042 1044 1046 1048 1050 1052 1054 1056 H1H5761P 1058 1060 1062 1064 1066 1068 1070 1072 H1H5763P 1074 1076 1078 1080 1082 1084 1086 1088 H1H5764P 1090 1092 1094 1096 1098 1100 1102 1104 H1H5769P 1132 1134 1136 H1H5772P 1138 1140 1142 1144 1146 1148 1150 1152 H1H5777P 1154 1156 11581160 1162 1164 1166 1168 H1H5778P 1170 1172 1174 1176 1178 1180 1182 1184 H1H5780P 1186 1188 1190 1192 1194 1196 1198 1200 H1H5781P 1202 1204 1206 1208 1210 1212 1214 1216 H1H5782P 1218 1220 1222 1224 1226 1228 1230 1232 Petition 870260046565, dated 05 / 15 / 2026, page 193 / 435 185 / 202 SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H1H5785B 1234 1236 1238 1240 1242 1244 1246 1248 H1H5786B 1250 1252 1254 1256 1258 1260 1262 1264 H1H5788P 1266 1268 1270 1272 1274 1276 1278 1280 H1H5790B 1282 1284 1286 1288 1290 1292 1294 1296 H1H5791B 1298 1300 1302 1304 1306 1308 1310 1312 H1H5792B 1342 1344 H1H5795B 1346 1348 1350 1352 1354 1356 1358 1360 H1H5796B 1362 1364 1366 1368 1370 1372 1374 1376 H1H5797B 1378 1380 1382 1384 1386 1388 1390 1392 H1H5798B 1394 1396 1398 1400 1402 1404 1406 1408 H1H5799P 1410 1412 1414 1416 1418 1420 1422 1424 H1H5801B 1426 1428 1430 1432 1434 1436 1438 1440 H1H7194B 1442 1444 1446 1448 1634 1636 1638 1640 H1H7195B 1450 1452 1454 1456 1634 1636 1638 1640 H1H7196B 1458 1460 1462 1464 1634 1636 1638 1640 H1H7198B 1466 1468 1470 1472 1634 1636 1638 1640 H1H7203B 1474 1476 1478 1480 1634 1636 1638 1640 H1H7204B 1482 1484 1486 1488 1634 16361638 1640 H1H7208B 1490 1492 1494 1496 1634 1636 1638 1640 H1H7211B 1498 1500 1502 1504 1634 1636 1638 1640 H1H7221B 1506 1508 1510 1512 1634 1636 1638 1640 H1H7223B 1514 1516 1518 1520 1634 1636 1638 1640 H1H7226B 1522 1524 1526 1528 1634 1636 1638 1640 H1H7232B 1530 1532 1534 1536 1634 1636 1638 1640 Petition 870260046565, dated 05 / 15 / 2026, page 194 / 435 186 / 202 SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR3 LCVR LCDR11 LCDR2 LCDR3 H1H7233B 1538 1540 1542 1544 1634 1636 1638 1640 H1H7241B 1546 1548 1550 1552 1634 1636 1638 1640 H1H7242B 1554 1556 1558 1560 1634 1636 1638 1640 H1H7250B 1562 1564 1566 1568 1634 1636 1638 1640 H1H7251B 1570 1572 1574 1576 1634 1636 1638 1640 H1H7254B 1638 1640 H1H7269B 1594 1596 1598 1600 1634 1636 1638 1640 H1H7279B 1602 1604 1606 1608 1634 1636 1638 1640 H1xH7221G 1610 1612 1614 1616 1634 1636 1638 1640 H1xH7221G3 1618 1620 1622 1624 1634 1636 1638 1640 H1xH7221G5 1626 1628 1630 1632 1634 1636 1638 1640 TABLE 17: ACID SEQUENCE IDENTIFIERS NUCLEECE SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1M2712N 401 403 405 407 409 411 413 415 H1M2692N 417 419 421 423 425 427 429 431 H1M3542N 433 435 437 439 441 443 445 447 H1M3544N 449 451 453 455 457 459 461 463 H1M3549N 465 467 469 471 473 475 477 479 H1M3613N 481 483 485 487 489 491 493 495 H2M2689N 497 499 501 503 505 507 509 511 H2M2690N 513 515 517 519 521 523 525 527 Petition 870260046565, dated 05 / 15 / 2026, page 195 / 435 187 / 202 SEQ ID NOs: Design of Antivirus HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H2M2691N 529 531 533 535 537 539 541 543 H2M2704N 545 547 549 551 553 555 557 559 H2M2705N 561 563 565 567 569 571 573 575 H2M2706N 577 579 581 583 585 587 589 591 H2M2707N 593 595 597 599 601 603 605 607 H2M2708N 609 611 613 615 617 619 621 623 H2M2709N 625 627 629 631 633 635 637 639 H2M2710N 641 643 645 647 649 651 653 655 H2M2711N 657 659 661 663 665 667 669 671 H2M2774N 673 675 677 679 681 683 685 687 H2M2775N 689 691 693 695 697 699 701 703 H2M2776N 705 707 709 711 713 715 717 719 H2M2777N 721 723 725 727 729 731 733 735 H2M2778N 737 739 741 743 745 747 749 751 H2M2779N 753 755 757 759 761 763 765 767 H2M2789N 769 771 773 775 777 779 781 783 H2M2862N 785 787 789 791 793 795 797 799 H2M2885N 801 803 805 807 809 811 813 815 H2M2886N 817 819 821 823 825 827 829 831 H2M3540N 833 835 837 839 841 843 845 847 H2M3541N 849 851 853 855 857 859 861 863 H2M3543N 865 867 869 871 873 875 877 879 H2M3547N 881 883885 887 889 891 893 895 H2M3548N 897 899 901 903 905 907 909 911 H2M3563N 913 915 917 919 921 923 925 927 Petition 870260046565, dated 05 / 15 / 2026, page 196 / 435 188 / 202 SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H5751P 929 931 933 935 937 939 941 943 H1H5752P 945 947 949 951 953 955 957 959 H1H5753B 961 963 965 967 969 971 973 975 H1H5754B 977 979 981 983 985 987 989 991 H1H5755B 993 995 997 999 1001 1003 1005 1007 H1H5756B 1009 1011 1013 1015 1017 1019 1021 1023 1045 1047 1049 1051 1053 1055 H1H5761P 1085 1087 H1H5764P 1089 1091 1093 1095 1097 1099 1101 1103 1125 1127 1129 1131 1133 1135 H1H5772P 1137 1139 1141 1143 1145 1147 1149 1151 H1H5777P 1153 1155 1157 1159 1161 1163 1165 1167 H1H5778P 1169 1171 1173 1175 1177 1179 1181 1183 1205 1207 1209 1211 1213 1215 H1H5782P 1217 1219 1221 1223 1225 1227 1229 1231 H1H5785B 1233 1235 12371239 1241 1243 1245 1247 H1H5786B 1249 1251 1253 1255 1257 1259 1261 1263 H1H5788P 1265 1267 1269 1271 1273 1275 1277 1279 H1H5790B 1281 1283 1285 1287 1289 1291 1293 1295 H1H5791B 1297 1299 1301 1303 1305 1307 1309 1311 H1H5792B 1313 1315 1317 1319 1321 1323 1325 1327 Petition 870260046565, dated 05 / 15 / 2026, page 197 / 435 189 / 202 SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H5793B 1329 1331 1333 1335 1337 1339 1341 1343 H1H5795B 1345 1347 1349 1351 1353 1355 1357 1359 H1H5796B 1361 1363 1365 1367 1369 1371 1373 1375 H1H5797B 1377 1379 1381 1383 1385 1387 1389 1391 H1H5798B 1393 1395 1397 1399 1401 1403 1405 1407 H1H5799P 1435 1437 1439 H1H7194B H1H7196B 1457 1459 1461 1463 1633 1635 1637 1639 H1H7198B 1637 1639 H1H7204B 1481 1483 1485 1487 1633 1635 1637 1639 H1H7208B 1489 1491 1493 1495 1633 1635 1637 1639 H1H7211B 1497 1499 1501 1503 1633 1635 1637 1639 1505 1507 1509 1511 1633 1635 1637 1639 H1H7223B 1513 1515 1517 1519 1633 1635 1639 H1H7226B 1521 1523 1525 1527 1633 1635 1637 1639 H1H7232B 1529 1531 1533 1535 16331635 1637 1639 H1H7233B 1537 1539 1541 1543 1633 1635 1637 1639 H1H7241B 1545 1547 1549 1551 1633 1635 1637 1639 H1H7242B 1553 1555 1557 1559 1633 1635 1637 1639 1573 1575 1633 1635 1637 1639 H1H7254B 1577 1579 1581 1583 1633 1635 1637 1639 Petition 870260046565, dated 05 / 15 / 2026, page 198 / 435 190 / 202 SEQ ID Nos: Antibody Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H7258B 1585 1587 1589 1591 1633 1635 1637 1639 H1H7269B 1593 1595 1597 1599 1633 1635 1637 1639 H1H7279B 1601 1603 1605 1607 1633 1635 1637 1639 H1xH7221G 1609 1611 1613 1615 1633 1635 1637 1639 H1xH7221G3 1617 1619 1621 1623 1633 1635 1637 1639 H1xH7221G5 1625 1627 1629 1631 1633 1635 1637 1639

[00386] Antibodies are typically referred to in this document according to the following nomenclature: Fc prefix (e.g., H1H, H1M, H2M, etc.), followed by a numerical identifier (e.g., 2712, 2692, etc., as shown in Table 1), followed by a P, N, or B suffix. Therefore, according to this nomenclature, an antibody may be referred to in this document, for example, as H1H2712N, H1M2692N, H2M2689N, etc. The H1H, H1M, and H2M prefixes in the antibody designations used in this document indicate the particular Fc region isotype of the antibody. For example, an H1H antibody has a human IgG1 Fc pattern, an H1M antibody has a mouse IgG1 Fc pattern, and an H2M antibody has a mouse IgG2 Fc pattern (all variable regions are fully human as denoted by the first H in the antibody designation).As will be observed by a person of ordinary skill in the technique, an antibody that has a particular Fc isotype can be converted into an antibody with a different Fc isotype (for example, an antibody with a mouse IgG1 Fc can be converted into an antibody with a human IgG4, etc.), however, in any case, the variable domains (including the CDRs) – which are indicated by the numerical identifiers shown in Table 1 – will remain the same, and the properties of... Petition 870260046565, dated 05 / 15 / 2026, p. 199 / 435 191 / 202 linkage as expected so that they are identical or substantially similar regardless of the nature of the Fc domain.

[00387] Tables 18 and 19 present amino acid sequence identifiers for variable heavy chain regions (Table 18) and variable light chain regions (Table 19), and their corresponding CDRs, of additional anti-CD3 HCVRs and LCVRs useful in the anti-MUC16 x anti-CD3 bispecific antibodies of the invention. TABLE 18 (REGION AMINO ACID SEQUENCES) HEAVY CHAIN ​​VARIABLE SEQ ID Nos Heavy Chain Identifier HCVR HCDR1 HCDR2 HCDR3 CD3-VH-AA 1642 1644 1646 1648 CD3-VH-B 1658 1660 1662 1664 CD3-VH-C 1674 1676 1678 1680 CD3-VH-D 1690 1692 1694 1696 CD3-VH-E 1706 1708 1710 1712 CD3-VH-F# 1721 1722 1723 1724 TABLE 19 (REGION AMINO ACID SEQUENCES) (Light Chain Variable) SEQ ID Numbers Light Chain Identifiers LCVR LCDR1 LCDR2 LCDR3 CD3-VL-AA 1650 1652 1654 1656 CD3-VL-B 1666 1668 1670 1672 CD3-VL-C 1682 1684 1686 1688 CD3-VL-D 1698 1700 1702 1704 Petition 870260046565, dated 05 / 15 / 2026, pp. 200 / 435 192 / 202 SEQ ID Numbers Light Chain Identifiers LCVR LCDR1 LCDR2 LCDR3 CD3-VL-E 1714 1716 1718 1720 CD3-VL-F# 1725 1726 1727 1728

[00388] The variable heavy and light chain regions of CD3-VH-F and CD3-VL-F were derived from the anti-CD3 antibody designated L2K as presented in document WO2004 / 106380.

[00389] In addition, Tables 20 and 21 present the sequence identifiers for the nucleotide sequences encoding the heavy chain variable regions (Table 20) and the light chain variable regions (Table 21), and their corresponding CDRs, of additional anti-CD3 HCVRs and LCVRs useful in bispecific anti-MUC16 x anti-CD3 antibodies of the invention. TABLE 20 (NUCLEOTIDE SEQUENCES THAT ENCODE THE VARIABLE REGION HEAVY CHAIN ​​SEQUENCES) SEQ ID Nos Heavy Chain Identifier HCVR HCDR1 HCDR2 HCDR3 CD3-VH-AA 1641 1643 1645 1647 CD3-VH-B 1657 1659 1661 1663 CD3-VH-C 1673 1675 1677 1679 CD3-VH-D 1689 1691 1693 1695 CD3-VH-E 1705 1707 1709 1711 TABLE 21 (NUCLEOTIDE SEQUENCES THAT ENCODE THE VARIABLE REGION OF LIGHT CHAIN ​​SEQUENCES) Petition 870260046565, dated 05 / 15 / 2026, p. 201 / 435 193 / 202 SEQ ID Numbers Light Chain Identifiers LCVR LCDR1 LCDR2 LCDR3 CD3-VL-AA 1649 1651 1653 1655 CD3-VL-B 1665 1667 1669 1671 CD3-VL-C 1681 1683 1685 1687 CD3-VL-D 1697 1699 1701 1703 CD3-VL-E 1713 1715 1717 1719 CONTROL CONSTRUCTS USED IN THE FOLLOWING EXAMPLES

[00390] Several control constructs (anti-CD3 antibodies) were included in the following experiments for comparative purposes: OKT-3, a mouse monoclonal antibody against human T cell surface antigens available from the American Type Culture Collection (ATCC) under catalog number CRL-8001; and SP34, a commercially available mouse monoclonal antibody obtained, for example, from Biolegend, San Diego, CA (Catalog No.: 302914) or BD Pharmagen, Catalog No.: 55052, reactive against the epsilon chain of the T3 complex in human T lymphocyte cells. EXAMPLE 13: GENERATION OF ADDITIONAL A...

Claims

1. Bispecific antibody or antigen-binding fragment thereof, characterized in that it comprises a first antigen-binding domain that binds specifically to human CD3, and a second antigen-binding domain that binds specifically to human mucin 16 (MUC16), wherein the first antigen-binding domain that binds specifically to human CD3 comprises domains HCDR1-HCDR2-HCDR3-LCDR1LCDR2-LCDR3, respectively, comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 1732-1734-1736-28-30-32; 1764-1766-1768-28-30-32; 1780-1782-1784-28-30-32; 1788-1790-1792-28-30-32 and 1868-1870-1872-28-30-32, and the second antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32.

2. Bispecific antibody or antigen-binding fragment, according to claim 1, characterized in that the second antigen-binding domain that binds to human MUC16 comprises a variable heavy chain region comprising the amino acid sequence of SEQ ID NO: 18 and a variable light chain region comprising the amino acid sequence of SEQ ID NO:

26.

3. Bispecific antibody or antigen-binding fragment, according to claim 1, characterized in that the first antigen-binding domain that binds to human CD3 comprises a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1730; 1762; 1778; 1786; and 1866, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

26.

4. Bispecific antibody or antigen-binding fragment, according to claim 1, characterized in that the first antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 1732-1734-1736-28-30-32, and the second antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32.

5. Bispecific antibody or antigen-binding fragment, according to claim 1, characterized in that the first antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 1868-1870-1872-28-30-32, and the second antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32.

6. Bispecific antibody or antigen-binding fragment, according to claim 2, characterized in that the first antigen-binding domain that binds to human CD3 comprises a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1730; 1762; 1778; 1786; and 1866, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

26.

7. Bispecific antibody or antigen-binding fragment, according to claim 2, characterized in that the first antigen-binding domain comprises domains Petition 870260046565, dated 05 / 15 / 2026, page 213 / 435 3 / 5 HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 1732-1734-1736-28-30-32, and the second antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32.

8. Bispecific antibody or antigen-binding fragment, according to claim 2, characterized in that the first antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 1868-1870-1872-28-30-32, and the second antigen-binding domain comprises domains HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 20-22-24-28-30-32.

9. Bispecific antibody or antigen-binding fragment, according to claim 7, characterized in that the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1730 and an LCRV comprising the amino acid sequence of SEQ ID NO: 26, and in that the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 18 and an LCRV comprising the amino acid sequence of SEQ ID NO:

26.

10. Bispecific antibody or antigen-binding fragment, according to claim 8, characterized in that the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1866 and an LCRV comprising the amino acid sequence of SEQ ID NO: 26, and in that the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 18 and an LCRV comprising the amino acid sequence of SEQ ID NO:

26.

11. Bispecific antibody, according to claim 9, characterized in that it comprises a first binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1961 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960, and a second binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1959 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960.

12. Bispecific antibody, according to claim 10, characterized in that it comprises a first binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1962 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960, and a second binding domain comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 1959 and a light chain comprising the amino acid sequence of SEQ ID NO: 1960.

13. Pharmaceutical composition, characterized in that it comprises the bispecific antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 12, and a pharmaceutically acceptable vehicle or diluent.

14. Use of the bispecific antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 12, characterized in that it is for the manufacture of a medicament to treat a cancer that expresses MUC16.

15. Use, according to claim 14, characterized Petition 870260046565, dated 05 / 15 / 2026, page 215 / 435 5 / 5 by the fact that the cancer expressing MUC16 is selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, mass-forming type intrahepatic cholangiocarcinoma, cervical adenocarcinoma and gastric tract adenocarcinoma.

16. Use, according to claim 15, characterized in that the cancer expressing MUC16 is ovarian cancer.

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