CD3-Targeted T Cell Engagers with Improved Therapeutic Index

Abstract

Antibodies against CD3 complex have been tested in bispecific and trispecific antibody formats that further recognize tumor-associated antigens.However, such antibodies are generally accompanied by on-target toxicity.It is revealed herein that bispecific and trispecific antibodies can reduce or avoid toxicity by adopting suitable structures and using anti-CD3 units with suitable balanced T cell activation activity levels.Such bispecific and trispecific antibody structures are provided.A wide range of anti-CD3 antibodies with different T cell activation activity levels are also provided, which can be easily used in different scenarios.

Description

[Background technology]

[0001] background T cell engagers with specificity for T cells (e.g., CD3 bispecific antibodies) provide a targeted cancer immunity platform that binds the patient's own T cells to malignant cells. Such bispecific T cell engagers ensure direct binding of T cells to cancer cells, thus enabling T cell activation, resulting in cytotoxic activity being directed against the cancer cells.

[0002] There are now many bispecific T cell engagers with anti-CD3 moieties. These bispecific antibodies, with different molecular designs and binding properties, have been tested in preclinical and clinical development for the treatment of liquid or solid tumors. Although many of them have shown potent cancer cell killing in vitro, their development is often accompanied by safety concerns. These antibodies tend to cause excessive release of cytokines that can lead to potentially fatal cytokine release syndrome (CRS); target organ toxicity (off-tumor / on-target cytotoxicity) due to the redirection of T cells to normal tissues expressing tumor-associated antigens (TAA); and neurotoxicity in some cases. Another major obstacle for CD3-based bispecific T cell engagers is that these antibodies are usually associated with limited efficacy for multiple reasons, such as the lack of sufficient effector T cells in the tumor microenvironment. Summary of the Invention [Means for solving the problem]

[0003] Abstract The present disclosure provides a class of novel T cell engagers that have a greatly improved therapeutic index and significant T cell infiltration into the tumor site compared to conventional T cell engagement therapies, thereby enabling the treatment of difficult to treat tumors such as cold tumors. One example of such a T cell engager disclosed herein includes anti-CD3 and anti-4-1BB units that provide sufficient and persistent T cell activation signals, together with a tumor-associated antigen (TAA) recognition unit to redirect T cells to the tumor site.

[0004] Over the decades since the initial attempts to generate therapeutic antibodies targeting CD3, few have been successful. SP34 was the first widely used anti-CD3 agonist antibody, which can cross-react with cynomolgus monkey CD3. Also, a significant number of derivatives have been developed based on the original SP34 antibody. Nevertheless, incorporating the SP34 antibody or its derivatives into the scFv format has proven to be a major challenge. Given that scFvs are the most commonly used fragments for incorporation into bispecific or trispecific antibodies, this deficiency has significantly limited the clinical application of SP34. Another widely used anti-CD3 antibody is OKT3, which unfortunately does not have cross-reactivity with cynomolgus monkey CD3, and therefore its clinical use is hindered by the lack of suitable preclinical trial models.

[0005] Current therapeutic use of anti-CD3 antibodies often involves off-target toxicity due to CD3 activation in the absence of targeted tumor cells. Clinical efficacy is therefore hindered by dose-limiting toxicity. This challenge is further complicated by the limited availability of anti-CD3 antibodies with different activities / physical and chemical properties / safety profiles.

[0006] It is demonstrated herein that the toxicity of T cell engagers can be reduced or avoided by utilizing anti-CD3 units with suitably balanced levels of T cell activation activity. The present disclosure provides anti-CD3 antibodies and fragments with such balanced T cell activation activity, as well as bispecific and trispecific antibody format T cell engagers containing such anti-CD3 units.

[0007] From just three murine anti-CD3 antibodies, we were able to generate approximately 70 different humanized versions, which surprisingly represent a broad range of anti-CD3 antibodies with diverse levels of T cell activation activity.

[0008] The present inventors have also developed a standardized assay for measuring T cell activation activity.In one example, T cell activation activity is measured by NFAT (nuclear factor of activated T cells) signaling assay, either as monospecific anti-CD3 antibody or bispecific antibody further comprising specificity for TAA.In NFAT assay, control antibody such as SP34 can be used as reference.In another example, T cell activation activity is measured for its ability to induce 4-1BB activity.

[0009] As shown in Table 10, any one of these assays or a combination of two or more of them can be used to categorize anti-CD3 antibodies into nine different grades (grades 1-9). Further testing surprisingly demonstrated that grade 3 and 4 anti-CD3 antibodies have well-balanced T cell activation activity suitable for the development of desired T cell engagers. As shown in Table 10, these grade 3-4 antibodies showed little or no CD3 agonism activity in standardized NFAT assays when tested as monospecific antibodies. More specifically, their T cell activation activity was less than 50% compared to SP34. Grade 5-9 antibodies, in contrast, are more potent (i.e., >50% SP34) as monospecific antibodies and therefore carry safety concerns.

[0010] Surprisingly, when tested in an anti-CD3 / anti-TAA bispecific format against TAA-expressing cells, grade 3-4 antibodies showed strong CD3 agonism activity (e.g., stronger than monospecific SP34). In contrast, grade 1-2 ones showed no detectable activity. Such moderate, well-balanced, CD3 agonism activity can also be measured by 4-1BB induction assay. As shown in Table 10, grade 3-4 anti-CD3 antibodies showed moderate (++ or +++) 4-1BB induction activity. In a preferred embodiment, the 4-1BB induction assay is performed using a trispecific antibody comprising an anti-CD3 unit, an anti-4-1BB unit, and an anti-TAA unit.

[0011] As demonstrated in the experimental examples, grade 3-4 anti-CD3 antibodies showed exceptional efficacy and therapeutic index when incorporated into bispecific and trispecific formats. Thus, these data demonstrate that anti-CD3 antibodies with well-balanced CD3 agonism activity may be excellent candidates for incorporation into bispecific and trispecific T cell engagers. Also importantly, anti-CD3 antibodies of different potencies may be suitable for different scenarios. Therefore, the antibodies obtained here may be suitable for a wide range of clinical uses, thus enabling application in off-the-shelf immune-oncology treatments.

[0012] Another advantage of the present technology is that, unlike SP34, the anti-CD3 antibodies of the present disclosure have excellent development potential because they can be easily generated in scFv format and engineered to become bispecific and trispecific antibodies.

[0013] Various embodiments of bispecific and trispecific antibodies of the present disclosure include an anti-4-1BB unit. 4-1BB is a vital costimulatory signal that plays a key role in T cell activation and proliferation, with the added benefit of anti-exhaustion and memory. Trispecific antibodies that simultaneously target TAA, CD3 and 4-1BB, as we demonstrate herein, engage T cells with two signals, CD3 (first TCR signal) and 41BB (second costimulatory signal), resulting in sustained and efficient activation of T cells. Furthermore, CD3 signals can synergize 4-1BB signals by inducing 4-1BB expression on T cells. Such synergy is further enhanced when the anti-4-1BB unit is a tumor-associated antigen (TAA)-dependent agonist antibody that does not activate 4-1BB signaling in the absence of TAA-expressing cells.

[0014] Surprisingly, the synergistic effect of CD3 signaling and 4-1BB signaling is even more evident when the anti-CD3 unit and the anti-4-1BB unit are constructed into one antibody. The TAA-CD3-4-1BB trispecific antibody showed greater T cell activation compared to the combination of the TAA-CD3 and TAA-4-1BB bispecific antibodies. This suggests a unique advantage of the TAA-CD3-4-1BB trispecific format.

[0015] Thus, one embodiment of the present disclosure provides a multispecific antibody comprising an anti-CD3 unit comprising an anti-CD3 antibody or antigen-binding fragment having binding specificity for human CD3 complex; and an anti-4-1BB unit comprising an anti-4-1BB antibody or antigen-binding fragment having binding specificity for human 4-1BB protein. In some embodiments, the multispecific antibody further comprises an anti-tumor associated antigen (TAA) unit comprising an anti-TAA antibody or antigen-binding fragment having binding specificity for human TAA.

[0016] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment is a tumor-associated antigen-dependent agonist antibody or antigen-binding fragment. In some embodiments, the anti-4-1BB antibody or antigen-binding fragment binds to the cysteine-rich domain 2 (CRD2), CRD3 or CRD4 of the extracellular domain of 4-1BB protein. In some embodiments, the anti-4-1BB antibody or antigen-binding fragment binds to the CRD3 or CRD4 of the extracellular domain of 4-1BB protein.

[0017] In some embodiments, anti-CD3 unit has a balanced T cell activation activity. Methods and standards for determining such balanced T cell activation activity are also described herein. In one example, T cell activation activity is measured by NFAT assay as maximum effect (Emax, which is the highest luminescence measurement from the assay). For each anti-CD3 antibody or fragment, two types of T cell activation activity (Emax) can be obtained, which are TAA-free and TAA-dependent.

[0018] In one embodiment, an anti-CD3 unit with balanced T cell activation activity has 50% or less of a non-TAA T cell activation activity compared to the monospecific anti-CD3 antibody SP34, where the non-TAA T cell activation activity indicates the activity of the anti-CD3 antibody to induce activation of T cells in a sample in the absence of the anti-CD3 antibody binding to TAA-expressing cells in the sample.

[0019] In one embodiment, an anti-CD3 unit having a balanced T cell activation activity has a TAA-dependent T cell activation activity that is greater than the TAA-free T cell activation activity of the SP34 antibody, and TAA-dependent T cell activation activity refers to the activity of an anti-CD3 antibody to induce activation of T cells in a sample in the presence of an anti-CD3 antibody further comprising an anti-TAA unit that binds to TAA-expressing cells in the sample.

[0020] In some embodiments, TAA-free or TAA-dependent T cell activation activity in response to an anti-CD3 antibody was measured by administering an anti-CD3 antibody at a concentration ranging from 1 nM to 100 nM for 6 hours to 2.5×10 4 and measuring the activity of the reporter gene. In measuring the TAA-dependent T cell activation activity, in some embodiments, Jurkat T cells are incubated with 2.5×10 4 The cells are mixed with TAA-expressing cells.

[0021] In some embodiments, the T cell activation activity of the antibody is measured by 4-1BB induction assay. In one embodiment, the multispecific antibody has a TAA-free 4-1BB induction rate of 5%, 10%, 15%, 20% or 25% or less at 100 nM, and the TAA-free 4-1BB induction rate is measured by incubating human peripheral blood CD8+ cells with the multispecific antibody at a final concentration of 100 nM for 48 hours at 37°C in the absence of TAA-expressing cells; and measuring the percentage of 4-1BB+CD8+ cells relative to the total number of CD8+ cells as the TAA-free 4-1BB induction rate. In some embodiments, the multispecific antibody is a trispecific antibody.

[0022] In some embodiments, the multispecific antibody has a TAA-dependent 4-1BB induction rate of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% at 1 nM, as measured by incubating a 1:1 ratio of human peripheral blood CD8+ cells and TAA-expressing cells with the multispecific antibody at a final concentration of 1 nM for 48 hours at 37° C.; and measuring the percentage of 4-1BB+CD8+ cells relative to the total number of CD8+ cells as the TAA-dependent 4-1BB induction rate. In some embodiments, the multispecific antibody is a trispecific antibody.

[0023] Therefore, it is important to identify anti-CD3 units with balanced potency for TAA-CD3 / 4-1BB trispecific T cell engagers that can better synergize with 4-1BB signals. A suitable anti-CD3 unit can efficiently activate T cells in the presence of tumor antigens, but cannot activate T cells in the absence of tumor antigens, as evidenced by, for example, IL2 activation, 4-1BB induction, and killing of TAA-positive target cells. With such a suitable anti-CD3 unit, a trispecific T cell engager can have strong 4-1BB activation potency, T cell activation, and tumor lethality in the presence of the corresponding TAA. Meanwhile, in the absence of the corresponding TAA, such a trispecific T cell engager has low (e.g., <10% 4-1BB-positive cells) or no 4-1BB induction potency, and no T cell activation and tumor lethality. Surprisingly, the TAA-CD3 / 4-1BB trispecific antibody can significantly inhibit over 90% of PD-1-resistant tumors; T cell infiltration, activation and proliferation without significant peripheral T cell activation. Moreover, unlike other T cell engagers: TAA-CD3, TAA-4-1BB and TAA-CD28, the TAA-CD3 / 4-1BB trispecific T cell engager can induce potent activation and infiltration of both CD4 and CD8 T cells into tumors.

[0024] In some embodiments, the anti-TAA unit comprises two anti-TAA Fabs fused via their respective heavy chains to the N-terminus of the Fc fragment, and the anti-CD3 unit comprises two scFvs each fused to the C-terminus of the light chain of the Fab of the anti-TAA unit.

[0025] In one embodiment, a multispecific antibody is also provided, comprising an anti-CD3 unit and an anti-tumor associated antigen (TAA) unit, wherein the anti-CD3 unit has binding specificity for human CD3 complex and has balanced CD3 agonist activity. The balanced CD3 agonist activity can be determined by the above-mentioned methods and criteria.

[0026] In some embodiments, the anti-CD3 unit comprises a single chain fragment (scFv) specific for the CD3 complex, In some embodiments, the anti-TAA unit comprises a Fab fragment comprising a pair of a heavy chain variable region (VH) and a light chain variable region (VL).

[0027] In some embodiments, the multispecific antibody further comprises an anti-4-1BB unit having specificity for human 4-1BB protein. In some embodiments, the anti-CD3 unit and the anti-TAA unit are both located at the N-terminus of the Fc fragment, and the anti-4-1BB unit is located at the C-terminus of the Fc fragment. In some embodiments, the anti-4-1BB unit comprises two nanobodies specific for human 4-1BB protein.

[0028] In some embodiments, the anti-4-1BB antibody or antigen-binding fragment is a tumor-associated antigen-dependent agonist antibody or antigen-binding fragment. In some embodiments, the anti-4-1BB antibody or antigen-binding fragment binds to the cysteine-rich domain 2 (CRD2), CRD3 or CRD4 of the extracellular domain of 4-1BB protein. In some embodiments, the anti-4-1BB antibody or antigen-binding fragment binds to the CRD3 or CRD4 of the extracellular domain of 4-1BB protein.

[0029] In some embodiments, the TAA is selected from the group consisting of claudin 18.2, 5T4, GPC3, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin. In some embodiments, the TAA is claudin 18.2.

[0030] In one embodiment, a multispecific antibody is also provided, comprising an anti-CD3 unit and an anti-4-1BB unit, wherein the anti-CD3 unit has binding specificity for human CD3 complex and has balanced CD3 agonist activity. In some embodiments, the anti-CD3 unit has a balanced 4-1BB induction ratio. The balanced CD3 agonist activity can be determined by the above-mentioned method and criteria.

[0031] In some embodiments, the anti-CD3 unit comprises a single chain fragment (scFv) specific for the CD3 complex. In some embodiments, the anti-CD3 unit is located at the N-terminus of the Fc fragment and the anti-4-1BB unit is located at the C-terminus of the Fc fragment. In some embodiments, the anti-4-1BB unit comprises two nanobodies specific for human 4-1BB protein. In some embodiments, the anti-4-1BB antibody or antigen-binding fragment is a tumor-associated antigen-dependent agonist antibody or antigen-binding fragment.

[0032] In some embodiments, the anti-CD3 unit comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2 and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2 and a VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO:7, VH CDR2 comprises the amino acid sequence of SEQ ID NO:8, 64, 65 or 66, VH CDR3 comprises the amino acid sequence of SEQ ID NO:9, VL CDR1 comprises the amino acid sequence of SEQ ID NO:10, VL CDR2 comprises the amino acid sequence of SEQ ID NO:11 and VL CDR3 comprises the amino acid sequence of SEQ ID NO:12 or 67; or; VH CDR1 comprises the amino acid sequence of SEQ ID NO:13, VH CDR2 comprises the amino acid sequence of SEQ ID NO:14, 68, 69 or 70, VH CDR3 comprises the amino acid sequence of SEQ ID NO:15, VL CDR1 comprises the amino acid sequence of SEQ ID NO:16 and VL CDR3 comprises the amino acid sequence of SEQ ID NO:17. CDR2 comprises the amino acid sequence of SEQ ID NO: 11 and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 17 or 71; or VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13 and VH CDR2 comprises the amino acid sequence of SEQ ID NO: 18, 72, 73 or 74 and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 19 and VL CDR1 comprises the amino acid sequence of SEQ ID NO: 20 and VL CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 75 and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or 76.

[0033] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 7, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 8, 64, 65 or 66, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 9, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 10, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 12 or 67.

[0034] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-28, optionally with a G55A substitution, an S61A substitution, or a combination thereof, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 30-32, optionally with a S93A substitution, wherein all amino acid positions are according to Kabat numbering.

[0035] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:24 and the VL comprises the amino acid sequence of SEQ ID NO:30; the VH comprises the amino acid sequence of SEQ ID NO:25 and the VL comprises the amino acid sequence of SEQ ID NO:30; or the VH comprises the amino acid sequence of SEQ ID NO:26 and the VL comprises the amino acid sequence of SEQ ID NO:30.

[0036] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 14, 68, 69 or 70, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 15, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 16, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 11 and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 17 or 71.

[0037] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 36-41, optionally with a G55A substitution, an S61A substitution, or a combination thereof, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 44-46, optionally with a S93A substitution, wherein all amino acid positions are according to Kabat numbering.

[0038] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:45; the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:45; the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:45; the VH comprises the amino acid sequence of SEQ ID NO:37 and the VL comprises the amino acid sequence of SEQ ID NO:46; the VH comprises the amino acid sequence of SEQ ID NO:38 and the VL comprises the amino acid sequence of SEQ ID NO:46; or the VH comprises the amino acid sequence of SEQ ID NO:39 and the VL comprises the amino acid sequence of SEQ ID NO:46.

[0039] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 37, optionally with a G55A substitution, an S61A substitution, or a combination thereof, and the VL comprises the amino acid sequence of SEQ ID NO: 45, optionally with a S93A substitution, wherein all amino acid positions are according to Kabat numbering.

[0040] In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 18, 72, 73 or 74, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 19, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 20, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 75, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or 76.

[0041] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 50-55, optionally having a G55A substitution, an S61A substitution, or a combination thereof, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 59-62, optionally having an S53A substitution, an S93A substitution, or a combination thereof, where all amino acid positions are according to Kabat numbering.

[0042] In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO:50 and the VL comprises the amino acid sequence of SEQ ID NO:60; or the VH comprises the amino acid sequence of SEQ ID NO:50 and the VL comprises the amino acid sequence of SEQ ID NO:61.

[0043] Methods and uses for using the disclosed antibodies or fragments to treat diseases such as cancer are also provided. In some embodiments, the cancer is a non-immunogenic tumor. In some embodiments, the non-immunogenic tumor is not infiltrated by T cells or is deficient in T cell infiltration or T cell activation. In some embodiments, the cancer is resistant to treatment with immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor is a PD-L1, PD-1 or CTLA-4 inhibitor, or a combination thereof.

[0044] In some embodiments, the cancer is prostate cancer, pancreatic cancer, or leukemia. In some embodiments, the cancer is breast cancer, colon cancer, gastric cancer, head and neck cancer, liver cancer, esophageal cancer, cervical cancer, thyroid cancer, lung cancer, bladder cancer, kidney cancer, uterine cancer, or melanoma. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 shows that anti-CD3 chimeric antibodies bound to human and cynomolgus monkey PBMCs with different potencies.

[0046] [Diagram 2] FIG. 2 shows that the CD3 activity of the tested chimeric antibodies gave a weaker response compared to the benchmark antibody OKT3.

[0047] [Diagram 3] FIG. 3 shows 4-1BB expression induced by the anti-CD3 chimeric antibodies tested.

[0048] [Figure 4] FIG. 4 shows the cell-based binding of the humanized CD3 antibodies tested.

[0049] [Figure 5-1] FIG. 5 shows that the humanized CD3 antibodies tested exhibited a wide variety of CD3 activities. [Figure 5-2] Same as above.

[0050] [Figure 6] FIG. 6 shows 4-1BB expression induced by humanized CD3 antibody.

[0051] [Figure 7] FIG. 7 shows CD3 NFAT activity examined by TCR / CD3 cells.

[0052] [Figure 8] FIG. 8 shows the TAA-dependent agonist activity of CD3 BsAb in comparison to SP34.

[0053] [Figure 9] FIG. 9 shows that bispecific antibodies in a 1+1 format induced a more robust response than bispecific antibodies in a 2+Lc2 format.

[0054] [Figure 10] FIG. 10 shows that 4-1BB-induced IL-2 secretion was CD3-dependent.

[0055] [Figure 11] FIG. 11 shows that trispecific antibody stimulated IL-2 secretion was positively correlated with CD3 NFAT activity.

[0056] [Figure 12] FIG. 12 shows that trispecific antibody stimulated 4-1BB induction positively correlates with CD3 NFAT activity.

[0057] [Figure 13] FIG. 13 shows IL-2 secretion and cytolytic activity induced by benchmark bispecific antibodies.

[0058] [Figure 14] FIG. 14 shows IL-2 secretion and cytolytic activity induced by benchmark trispecific antibodies.

[0059] [Figure 15] FIG. 15 shows the design and results of an in vivo experiment for treating B16F10-h5T4-induced tumors with trispecific antibodies.

[0060] [Figure 16] FIG. 16 shows the in vitro and in vivo effects of 5T4-CD3-4-1BB with different 5T4 binding epitopes.

[0061] [Figure 17] FIG. 17 shows the in vitro and in vivo effects of 5T4-CD3-4-1BB within 5T4 binding epitope D.

[0062] [Figure 18] FIG. 18 shows the in vitro and in vivo effects of 5T4-CD3-4-1BB with different CD3 activities.

[0063] [Figure 19-1] FIG. 19 displays ex vivo tumor immunophenotyping (IPT) analysis of the trispecific candidate antibodies tested. [Figure 19-2] Same as above.

[0064] [Figure 20] FIG. 20 shows ex vivo blood immunophenotyping (IPT) analysis of the trispecific candidate antibodies tested.

[0065] [Figure 21] FIG. 21 shows the results of cytokine release in the trispecific antibody treatment groups tested.

[0066] [Figure 22] FIG. 22 shows the advantage of the trispecific antibody over combination therapy in terms of IL-2 secretion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Detailed Description definition It should be noted that the term "a" or "an" entity refers to one or more of that entity, e.g., "an antibody" is understood to represent one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0068] As used herein, the term "polypeptide" includes the singular as well as the plural "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide," and the term "polypeptide" may be used in place of or synonymously with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modifications with non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a depicted nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0069] The term "isolated" as used herein with respect to a cell, a nucleic acid, e.g., DNA or RNA, refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, and culture medium, if produced by recombinant DNA techniques, or substantially free of chemical precursors and other chemicals, if chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not naturally occur as fragments and are not found in the natural state. The term "isolated" is also used herein to refer to a cell or polypeptide that is isolated from other cellular proteins or tissues. Isolated polypeptides are intended to encompass both purified and recombinant polypeptides.

[0070] As used herein, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen-binding fragment or single chain. Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein.

[0071] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds with the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0072] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and the variable region of the light chain (V L In some embodiments, the domains are linked by a short linker peptide of 10 to about 25 amino acids. The linker can contain many amino acids, such as glycine for flexibility, as well as serine or threonine for solubility, and the V H N-terminus of V L The scFv molecule can be linked either to the C-terminus of the IgG1A or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0073] The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with some subclasses within these (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to provide functional specialization. Modified versions of each of these classes and isotypes are readily discernible to one of skill in the art in light of this disclosure and, therefore, are within the scope of this disclosure. All immunoglobulin classes are clearly within the scope of this disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light polypeptide chains of molecular weight approximately 23,000 daltons, and two identical heavy polypeptide chains of molecular weight 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains flanking the heavy chains, starting at the mouth of the "Y" and continuing through the variable region.

[0074] Antibodies, antigen-binding polypeptides thereof, variants or derivatives of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments comprising the VK or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, the anti-CD3 antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0075] Light chains are classified as either kappa or lambda (K or λ). Each heavy chain may be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. For the heavy chains, the amino acid sequences run from the N-terminus at the forked ends of the Y to the C-terminus at the bottom of each chain.

[0076] Both the light and heavy chains are divided into structural and functional homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VK) and heavy (VH) chains determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and the constant domains of the heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, the C-terminal portion has the constant region, and the CH3 and CK domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0077] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK domain and the VH domain, or a subset of the complementarity determining regions (CDRs) of an antibody, combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) on each of the VH and VK chains. In some cases, for example, certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule may consist of only heavy chains, without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0078] In the case of naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional shape in an aqueous environment. The remainder of the amino acids in the antigen-binding domain, called the "framework" regions, show little intermolecular variability. The framework regions adopt a predominantly β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that allows for the positioning of the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids that respectively constitute the CDRs and framework regions can be easily identified for any given heavy or light chain variable region by those skilled in the art. because they have been precisely defined (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).

[0079] Where there are two or more definitions for a term used and / or accepted in the art, the definition of the term as used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the discontinuous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. MoI. Biol. 196:901-917 (1987), which references are incorporated herein by reference in their entirety. The CDR definitions by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. However, application of either definition to refer to the CDRs of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues that encompass the CDRs as defined by each of the above cited references are shown in the table below for comparison. The exact residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of an antibody. [Table 12]

[0080] Kabat et al. also define a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0081] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at approximately the 15th residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the 33rd amino acid residue after the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., after the cysteine ​​residue), includes approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins at approximately the 16th residue after the end of CDR-L1, and includes approximately 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), contains approximately 7-11 residues, and ends with the sequence F or WGXG, where X is any amino acid.

[0082] The antibody disclosed herein can be of any animal origin, including birds and mammals.Preferably, the antibody is human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody.In another embodiment, the variable region can be of chondrichthyan origin (e.g., from shark).

[0083] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region includes at least one of a CH1 domain, a hinge (e.g., upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may include a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain, a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure includes a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As indicated above, it will be understood by those skilled in the art that the heavy chain constant regions can be altered such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0084] The heavy chain constant region of the antibody disclosed herein can be derived from different immunoglobulin molecules.For example, the heavy chain constant region of the polypeptide can comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.In another example, the heavy chain constant region can comprise a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule.In another example, the heavy chain portion can comprise a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0085] As used herein, the term "light chain constant region" includes amino acid sequences derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0086] "Specifically binds" or "has specificity for" generally means that an antibody binds to an epitope through its antigen-binding domain, and that binding requires a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with a higher specificity than it has for related epitope "D."

[0087] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or alleviation of disease symptoms, and remission (whether partial or complete remission). "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in whom a condition or disorder is to be prevented.

[0088] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, farm animals, livestock, zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

[0089] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, e.g., mammalian subjects, who will benefit from the administration of an antibody or composition of the disclosure for use, e.g., in detection, diagnostic procedures, and / or treatment. T cell engagers with improved therapeutic index

[0090] CD3 (cluster of differentiation 3) is a protein complex that is involved in the differentiation of cytotoxic T cells (CD8 + Naive T cells) and helper T cells (CD4 + CD3 is a T cell coreceptor involved in activating both T-cells (immune T cells) and immune cells (naive T cells). CD3 is composed of four separate chains. In mammals, the complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and CD3-zeta (ζ chain) to generate an activation signal in T lymphocytes. The TCR, CD3-zeta, and other CD3 molecules together constitute the TCR complex.

[0091] Given their complex extracellular structure, anti-CD3 agonist antibodies are difficult to develop. Over the decades since the first attempts to generate therapeutic antibodies targeting CD3, few have been successful. SP34 was the first widely used anti-CD3 agonist antibody, which can cross-react with cynomolgus monkey CD3. Also, a significant number of derivatives have been developed based on the original SP34 antibody. Nevertheless, incorporating the SP34 antibody or its derivatives into the scFv format has proven to be a major challenge. Given that svFc is the most commonly used fragment for incorporation into bispecific or trispecific antibodies, this deficiency has significantly limited the clinical application of SP34. Another widely used anti-CD3 antibody is OKT3, which unfortunately does not have cross-reactivity with cynomolgus monkey CD3, and therefore its clinical use is hindered by the lack of suitable preclinical trial models.

[0092] Current therapeutic use of anti-CD3 antibodies often involves off-target toxicity due to CD3 activation in the absence of targeted tumor cells. This challenge is further complicated by the limited availability of anti-CD3 antibodies with different activities / physical and chemical properties / safety profiles.

[0093] In this regard, in one embodiment, the present disclosure provides new anti-CD3 antibodies, including humanized ones, and antigen-binding fragments thereof. Three mouse hybridoma clones, 153A6B1, 155A9B1 and 192A7B9 (Table 1), were generated. Humanization yielded between two and six humanized versions of each of the VH / VL (Tables 4-6). Combinations of these VH / VL sequences led to the production of less than 70 antibodies (Tables 4A-6A).

[0094] Surprisingly, these approximately 70 antibodies displayed very different in vitro functional activities as measured by nuclear factor of activated T cells (NFAT) luciferase reporter assay or 4-1BB induction assay. We categorized these antibodies into 9 different grades (Table 10, grades 1-9) based on NFAT activity or 4-1BB induction activity.

[0095] Some of these anti-CD3 antibodies were used to construct bispecific and trispecific antibodies (bispecific or trispecific T cell engagers). T cells require two signals to be fully activated. The first signal, which is antigen-specific, is provided by the T cell receptor (TCR) interacting with peptide-MHC molecules on the membrane of the antigen-presenting cell (APC). The second signal, the costimulatory signal, is antigen-nonspecific and is provided by the interaction of costimulatory molecules expressed on the membrane of the APC with costimulatory molecules expressed on the T cell. 4-1BB (CD137, tumor necrosis factor receptor superfamily 9), a member of the TNF-receptor superfamily (TNFRSF), is an important costimulatory molecule expressed after activation of immune cells, both innate and adaptive immune cells.

[0096] It is believed that a trispecific T cell engager with specificity for CD3, tumor-associated antigen (TAA) and 4-1BB can have at least the following advantages. First, such a trispecific antibody can simultaneously provide the first (TCR signal) and second (costimulatory) activation signaling to fully, persistently and efficiently activate T cells and further shape memory T cell responses, thus confronting malignant cells. Second, 41BB is an inducible costimulatory molecule expressed on activated or tumor-infiltrating T and NK cells. CD3 signal can further synergize with 41BB signal by inducing 4-1BB expression on T cells. Third, when a TAA-dependent anti-4-1BB antibody (i.e., an anti-4-1BB antibody that has no agonistic activity in the absence of the corresponding TAA) is used, the trispecific T cell engager becomes much safer in a non-tumor environment. Finally, the CD3 activity in the trispecific antibody can be adjusted (e.g., by using an appropriate grade of anti-CD3 moiety, e.g., Table 10) to fine-tune 4-1BB activation. Similarly, anti-CD3 antibodies of different potencies can be used as needed in bispecific T cell engagers that additionally target tumor-associated antigens (TAA).

[0097] Therefore, for such trispecific T cell engagers, it is important to identify anti-CD3 units with balanced potency that can better synergize with 4-1BB signals. A suitable anti-CD3 unit can efficiently activate T cells in the presence of tumor antigens, as evidenced by, for example, IL2 activation and 4-1BB induction, but cannot activate T cells in the absence of tumor antigens. With such a suitable anti-CD3 unit, the trispecific T cell engager can have strong 4-1BB activation potency in the presence of corresponding TAA, and can have low (e.g., <10% 4-1BB positive cells) or no 4-1BB activation potency in the absence of corresponding TAA.

[0098] Anti-CD3 / anti-4-1BB / anti-TAA trispecific antibodies with grade 4 antibodies (e.g., Hu153A6B1-2, -3 and -4, Hu155A9B1-8, -9, -10, -14, -15 and -16, and Hu192A7B9-7 and -13) were demonstrated to induce sufficient cytokine release and 4-1BB induction in TAA-positive cells without non-specific activation in control cells. Similarly, grade 3 antibodies (e.g., Hu153A6B1-7 and 13, Hu155A9B1-2, -3 and -4, and Hu192A7B9-4) also induced acceptable cytokine release and 4-1BB induction in TAA-positive cells without non-specific activation in TAA-negative control cells.

[0099] This disclosure also tested 20 newly developed anti-5T4 antibodies. Based on binding to 5T4, these antibodies were categorized into four binding groups, Bins A-D (Table 11), along with the benchmark antibody naptumomab. While naptumomab and some of the new antibodies belong to Bin A, Bin D antibodies were demonstrated to have the best performance, including when used in T cell engagers (Figure 16).

[0100] Furthermore, as shown in Figure 18, treatment with the trispecific 5T4-targeted T cell engager antibody significantly induced lymphocytic infiltration and expansion of T cell subtypes at the tumor site. Also, unlike other 4-1BB-based T cell engagers, treatment with the trispecific 5T4-targeted T cell engager antibody significantly induced lymphocytic infiltration and expansion of T cell subtypes at the tumor site. + Cells and CD8 + Both T cells were significantly increased, whereas NK cells remained unchanged. In addition, increased expression of the activation marker CD25 / costimulatory marker 4-1BB and proliferation marker Ki67 was observed in the tumor CD8 + T cells, suggesting a pro-inflammatory phenotype in the tumor microenvironment. + Helper T cell populations and CD8 +Effective boosting of both CTL cell populations is important for effective and long-term antitumor immune responses (Nature Reviews Immunology. 201:635-647). Meanwhile, treatment (with appropriate anti-CD3 units with balanced activity as further explained below) induced only slight activation of CD4 or CD8 cells in peripheral blood, indicating that these 5T4 / CD3 / 4-1BB trispecific antibodies have negligible impact on peripheral immune activation (Figure 19). Also, importantly, treatment did not show a significant increase in cytokine release (Figure 20). This highlights the safety of the present technology.

[0101] Based on this, these well-balanced anti-CD3 antibodies were subjected to further testing, and they showed the best efficacy and therapeutic index when incorporated into bispecific and trispecific formats. Thus, these data demonstrate that anti-CD3 antibodies with well-balanced T cell activation activity may be excellent candidates for incorporation into bispecific and trispecific T cell engagers. Also importantly, anti-CD3 antibodies with different potencies may be suitable for different scenarios. Therefore, the antibodies obtained now may be suitable for broad clinical use, thus enabling application in off-the-shelf immune-oncology therapy.

[0102] Furthermore, different structures of bi- and trispecific antibodies were tested. One of them, the 1+1 format, contains the anti-CD3 unit in a single chain fragment (scFv) and the anti-TAA unit in a regular Fab (VH / VL) domain. The other structure, the 2+Lc2 format, is symmetric and contains two anti-CD3 scFvs fused to the C-terminus of each of the two light chains of a pair of Fabs with specificity for TAA. For trispecific antibodies with additional specificity for 4-1BB, the anti-4-1BB unit may contain two nanobodies fused to the C-terminus of each of the Fc domains of the anti-TAA / CD3 bispecific unit (see diagrams in Figures 9A and 11A). It is revealed herein that the 1+1 format has a better safety margin and is therefore preferred, but the format 2+Lc2 is nevertheless acceptable.

[0103] Thus, according to one embodiment of the present disclosure, there is provided a multispecific antibody comprising an anti-CD3 unit and an anti-tumor associated antigen (TAA) unit. The multispecific antibody may be, but is not limited to, bispecific (anti-CD3 and anti-TAA), trispecific (anti-CD3, anti-TAA and anti-4-1BB or another co-stimulatory factor), or tetraspecific. Anti-CD3 unit with balanced T cell activation activity

[0104] In a preferred embodiment, the T cell engager of the present disclosure comprises an anti-CD3 unit with "balanced T cell activation activity" (or simply "balanced potency"). Examples of balanced T cell activation activity are represented by grades 3 or 4, as demonstrated in the accompanying experimental examples and as further explained below.

[0105] The potency of anti-CD3 units (e.g., antibodies or fragments) can be measured by a variety of different assays known in the art in different formats. In some embodiments, the assay is a 4-1BB induction assay. In some embodiments, the assay is a T cell proliferation assay. In some embodiments, the assay is a NFAT (nuclear factor of activated T cells) signaling assay. AT cell activation NFAT assay

[0106] In one example, T cell activation activity is measured by NFAT (nuclear factor of activated T cells) assay. In such an assay, T cell activation activity can be expressed as the maximum effect (Emax, which is the highest luminescence measurement from the assay). An example of an NFAT assay is performed as follows: Anti-CD3 antibody or antigen-binding fragment (concentration range: 1 nM to 100 nM) is added to 2.5 x 10 IgG containing a reporter gene regulated by the NFAT response element (NFAT-RE). 4 Jurkat T cells (e.g., from Promega, CAT#J1601) are incubated for 6 hours. Expression of the reporter gene is measured as a luminescence reading, the maximum of which is recorded as Emax and used to represent the T cell activation activity of the anti-CD3 antibody or fragment.

[0107] For each anti-CD3 antibody or fragment, two Emax values ​​can be obtained: "TAA-dependent Emax" or "Emax (TAA+)In one of them, called "T-cell specificity test," the test antibody also contains an antibody unit targeting a tumor-associated antigen (TAA) and is measured when Jurkat T cells are mixed with TAA-expressing cells (e.g., CHO cells or other tumor cells). In some embodiments, the cell number ratio between T cells and TAA-expressing cells is 1:1, or alternatively, 1:2, 2:1, 1:3, 3:1, 1:4 or 4:1. In some embodiments, such antibodies are bispecific (CD3+TAA) antibodies. In some embodiments, such antibodies are trispecific (e.g., CD3+TAA+4-1BB) antibodies. Non-limiting examples of tumor antigens include claudin 18.2, 5T4, GPC3, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin. In some embodiments, the TAA is claudin 18.2. In some embodiments, the TAA is 5T4.

[0108] "TAA-free Emax" or "Emax (TAA-) A second Emax value, referred to as "Emax 1," is measured when the test antibody does not contain additional anti-TAA units, when the incubation does not contain TAA-expressing cells, or when the two TAAs are different.

[0109] To enhance cross-platform reproducibility, a reference anti-CD3 antibody can be used and the activity can be expressed as a percentage of that of the reference antibody. One example of a reference anti-CD3 antibody is SP34 in a conventional full-size, monospecific Fab format (see sequences in Table 2B, SEQ ID NOs: 78 and 79). Thus, the NFAT assay conditions (e.g., Jurkat T cell count) can be varied without affecting the final result (% of SP34 activity). In a preferred embodiment, for the measurement of TAA-free Emax, the test anti-CD3 antibody is bivalent and the reference antibody (e.g., SP34) is also bivalent. For example, both are of a conventional full-size Fab antibody format. In some embodiments, for the measurement of TAA-free Emax, the test anti-CD3 antibody and the reference antibody (e.g., SP34) are of the same format, e.g., full-size Fab antibody, scFv, or multispecific antibody. However, such a choice does not apply in some embodiments to the measurement of TAA-dependent Emax.

[0110] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments of the present disclosure have a balanced T cell activation activity as measured by an NFAT assay that meets the following criteria: (A) Emax (TAA-) <[SP34 Emax (TAA-) 50% of], and (B) Emax (TAA+) >[SP34 Emax (TAA-) In some embodiments, for criterion (A), Emax (TAA-) is the Emax of SP34 (TAA-) 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% of Emax, while in (B), (TAA+) >[SP34 Emax (TAA-) In some embodiments, for criterion (A), Emax (TAA-) is the Emax of SP34 (TAA-)higher than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or 20% but less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70% of the Emax, whereas in (B), (TAA+) >[SP34 Emax (TAA-) ]. B. Alternative T cell activation assays

[0111] In some embodiments, the T cell activation activity of the anti-CD3 unit can be measured for the unit, or for the whole multispecific antibody, in an NFAT assay such as that described in the Examples. In some embodiments, the activity is expressed as an EC50 from the NFAT assay.

[0112] In some embodiments, an EC50 greater than 10 μg / mL is obtained when measurements are made in the absence of cells expressing the TAA (EC50 TAA- ), which is considered to have a balanced T cell activation activity. In some embodiments, for an anti-CD3 antibody or fragment with a balanced T cell activation activity, the EC50 or EC50 TAA- is greater than 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL or 300 μg / mL. In some embodiments, the EC50 for anti-CD3 antibodies or fragments with balanced T cell activation activity is TAA- is less than 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 1000 μg / mL or 5000 μg / mL.

[0113] In some embodiments, the anti-CD3 unit (or the entire multispecific antibody) has a T cell activation activity, EC50, of greater than 10 nM. In some embodiments, measurements are made in the absence of cells expressing the TAA (EC50 TAA-In some embodiments, the EC50 or EC50 for anti-CD3 antibodies or fragments with balanced T cell activation activity is TAA- is greater than 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, 200 nM or 300 nM. In some embodiments, for anti-CD3 antibodies or fragments with balanced T cell activation activity, the EC TAA- is less than 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 1000 nM or 5000 nM.

[0114] In some embodiments, the anti-CD3 unit is one that is greater than or equal to the EC TAA- TAA-dependent T cell activation activity EC50 at most 5-fold lower than that of TAA+ In some embodiments, an antibody is considered to have balanced T cell activation if it has an EC TAA+ is EC50 TAA- at most 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, 1 / 13, 1 / 14, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 40, 1 / 50, 1 / 100, 1 / 200, 1 / 500, or 1 / 1000 of the original.

[0115] In some embodiments, the EC TAA+ When measuring, the cell number ratio between T cells and TAA expressing cells is 1:1, or alternatively, 1:2, 2:1, 1:3, 3:1, 1:4 or 4:1.

[0116] In some embodiments, the EC TAA- is less than 100 μg / mL, 90 μg / mL, 80 μg / mL, 70 μg / mL, 60 μg / mL, 50 μg / mL, 40 μg / mL, 30 μg / mL, 20 μg / mL, 15 μg / mL, 10 μg / mL, 9 μg / mL, 8 μg / mL, 7 μg / mL, 6 μg / mL, 5 μg / mL, 4 μg / mL, 3 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL or 0.1 μg / mL. TAA-is less than 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM or 0.1 nM. C.4-1BB induction assay

[0117] In some embodiments, the T cell activation activity of the anti-CD3 unit is measured by a 4-1BB induction assay. In one embodiment, the 4-1BB induction assay is performed on monoclonal / monospecific anti-CD3 antibodies in a conventional format.

[0118] The 4-1BB inducing activity of monoclonal / monospecific anti-CD3 antibodies in conventional format can be determined as follows: Primary human PBMCs were cultured at 1×10 cells per well. 5 Human PBMC cells can be cultured in 96-well plates at a density of approximately 30% CD8, which is optimal for the assay. + The plate generally contains cells. The antibodies to be tested can be serially diluted and added to a 96-well plate at a final concentration, starting, for example, from 20 nM. After 48 hours of incubation at 37° C., the cells can be harvested. After washing with PBS, the samples can be stained using standard procedures by incubation with the following antibodies: anti-human CD4-APC (Ebiosciene, 17-0048-42), anti-human CD8-BV510 (BD bioscience, 563919), anti-human 4-1BB-PE (BD Pharmingen, 555956) for 30 minutes at room temperature in the dark. The samples can then be washed twice with FACS buffer. After centrifugation, the supernatant is discarded and the cells are resuspended in 0.2 mL of FACS buffer. CD8 + 4-1BB in T cells + CD8 + T cell subsets can be assessed by MACSQuant Analyzer 16. Total CD8 + 4-1BB on T cells + CD8 +Use the percentage (%) of T cells to indicate 4-1BB induction:

number

[0119] More briefly, the 4-1BB-inducing activity of a monospecific anti-CD3 antibody is measured by incubating human peripheral blood CD8+ cells with a monospecific antibody containing anti-CD3 units, for example at a final concentration of 20 nM, for 48 hours at 37° C., and measuring the percentage of 4-1BB+CD8+ cells relative to the total number of CD8+ cells as the 4-1BB induction rate. In some embodiments, the monospecific anti-CD3 antibody is bivalent. In some embodiments, the monospecific anti-CD3 antibody has a conventional Fab+Fc format.

[0120] The final concentration of the antibody can be, but is not limited to, 20 nM as described above, or alternatively, 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, 50 nM or 100 nM. In a preferred embodiment, the concentration is 10 nM or 20 nM.

[0121] The above example of a 4-1BB-induced activity measurement procedure uses primary human peripheral blood cells from a donor individual. In some embodiments, alternative cells, such as CD8 from an established cell line, may be used. + Cells may be used.

[0122] Although the above 4-1BB induction assay is described to measure the T cell activation activity of monospecific anti-CD3 antibodies, it can also be used for bispecific or trispecific / multispecific antibodies. For bispecific or trispecific / multispecific antibodies that further comprise an anti-TAA unit, the 4-1BB induction activity can be TAA-dependent 4-1BB induction activity (e.g., co-incubation with TAA-expressing cells) or TAA-free 4-1BB induction activity (e.g., no co-incubation with TAA-expressing cells).

[0123] In some embodiments, these TAA-free or TAA-dependent 4-1BB induction rates are measured when the multispecific antibody does not have an anti-4-1BB unit. In some embodiments, these TAA-free or TAA-dependent 4-1BB induction rates are measured when the multispecific antibody further comprises an anti-4-1BB unit.

[0124] In some embodiments, the measurements are performed on a multispecific antibody in a 1+1 format. In some embodiments, the measurements are performed on a multispecific antibody in a 2+2Lc format. In some embodiments, the measurements are performed on a trispecific antibody of the present disclosure. In some embodiments, the trispecific antibody comprises an anti-CD3 unit, an anti-TAA unit, and an anti-4-1BB unit. In some embodiments, the anti-4-1BB unit comprises one or two TAA-dependent agonist antibodies or fragments.

[0125] The TAA-free and TAA-dependent 4-1BB induction rates of multispecific antibodies can be measured by methods similar to those for monospecific antibodies. For example, human PBMCs were cultured at 1 x 10 cells per well. 5 Target cell lines expressing TAA (TAA-expressing cells) or control cells (CHO-K1) can be cultured in 96-well plates at a density of 2.5×10 4 density (hence a 4:1 E:T ratio, or a 1:1 CD8 +The cells can be seeded at a 5% 5% CO2 ratio (5:T ratio). Test antibodies can be serially diluted and added to a 96-well plate at final concentrations starting at 100 nM. After 48 hours of incubation at 37° C., the cells can be harvested. After washing with PBS, the samples can be stained using standard procedures by incubation with the following antibodies: anti-human CD4-APC (Ebiosciene, 17-0048-42), anti-human CD8-BV510 (BD bioscience, 563919), anti-human 4-1BB-PE (BD Pharmingen, 555956) for 30 minutes at room temperature in the dark. The samples can be washed twice with FACS buffer. After centrifugation, the supernatant can be discarded and the human PBMCs can be resuspended in 0.2 mL of FACS buffer. CD8 + 4-1BB in T cells + CD8 + T cell subsets can be assessed by MACSQuant Analyzer 16. 4-1BB induction rate can be calculated as follows:

number

number

[0126] More briefly, in some embodiments, the TAA-free 4-1BB induction ratio is determined by incubating human peripheral blood CD8+ cells with a multispecific antibody at a final concentration of 100 nM for 48 hours at 37° C. in the absence of TAA-expressing cells, and measuring the ratio of 4-1BB to the total number of CD8+ cells. + CD8 + In some embodiments, the multispecific antibody has a TAA-dependent 4-1BB induction rate of at least 20% at 100 nM, and the TAA-dependent 4-1BB induction rate is measured by measuring the percentage of cells that are TAA-free 4-1BB induction rate at a 1:1 ratio of human peripheral blood CD8 +Incubation of cells and TAA-expressing cells with multispecific antibodies at a final concentration of 100 nM for 48 h at 37°C and 4-1BB on the total number of CD8+ cells + CD8 + It is measured by determining the percentage of cells that express TAA-dependent 4-1BB induction.

[0127] In some embodiments, the multispecific antibody has a significantly higher 4-1BB induction activity in the presence of TAA-expressing cells than in their absence. For example, in some embodiments, the multispecific antibody has a 4-1BB induction rate that is at least 2-fold compared to the non-TAA 4-1BB induction rate. In some embodiments, the multispecific antibody has a 4-1BB induction rate that is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or 20-fold compared to the non-TAA 4-1BB induction rate.

[0128] In some embodiments, the multispecific antibody is bispecific. In some embodiments, the multispecific antibody is trispecific. In some embodiments, the multispecific antibody has a trispecific format as shown in format 1+1 (Figure 9), in which the anti-TAA unit is a Fab fragment, the anti-CD3 unit is an scFv, and the anti-4-1BB unit comprises two nanobodies fused to the C-terminus of the Fc fragment.

[0129] In some embodiments, the anti-CD3 unit in the multispecific antibody comprises at least one single chain fragment (scFv). In some embodiments, the anti-CD3 unit in the multispecific antibody comprises only one scFv. In some embodiments, the scFv is fused to the N-terminus of Fc, optionally via a linker or hinge fragment.

[0130] In some embodiments, the anti-TAA unit in a multispecific antibody comprises a Fab fragment consisting of a VH / VL pair. In some embodiments, the anti-TAA unit is fused to the N-terminus of Fc, optionally via a linker or hinge fragment.

[0131] The Fc fragments of the multispecific antibodies are optionally modified to contain knobs-in-holes or modified salt bridges to reduce mispairing of asymmetric antibodies compared to wild-type human Fc fragments.

[0132] In some embodiments, the multispecific antibody further comprises an antigen-binding unit. In some embodiments, the additional antigen-binding unit has specificity for human 4-1BB protein. In some embodiments, the anti-4-1BB unit is fused to the N-terminus of one or both of the two Fc chains. In some embodiments, the anti-4-1BB unit comprises one or a pair of scFvs. In some embodiments, the anti-4-1BB unit comprises one or a pair of nanobodies. In some embodiments, the anti-4-1BB unit is a non-agonist anti-4-1BB antibody or a fragment thereof. A non-agonist anti-4-1BB antibody or fragment binds to but does not activate 4-1BB in the absence of binding to a tumor-associated antigen.

[0133] In some embodiments, anti-CD3 antibodies or fragments considered to have balanced T cell activation activity have a TAA-free 4-1BB induction rate that is between 1% and 25%. In some embodiments, the 4-1BB induction rate is less than or equal to 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. In some embodiments, the 4-1BB induction rate is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, or 25%.

[0134] In some embodiments, the TAA-free 4-1BB induction rate for anti-CD3 antibodies or fragments considered to have balanced T cell activation activity is between 1% and 25%, 1% and 24%, 1% and 23%, 1% and 22%, 1% and 21%, 1% and 20%, 1% and 19%, 1% and 18%, 1% and 17%, 1% and 16%, 1% and 15%, 1% and 14%, 1% and 13%, 1% and 12%, 1% and 11%, 1% and 10%, 2% and 25%, 2% and 24%, 2% and 23%, 2% and 22%, 2% and 21%, 2% and 20%, 2% and 19%, 2% and 18%, 2% and 17%, 2% and 16% , 2%~15%, 2%~14%, 2%~13%, 2%~12%, 2%~11%, 2%~10%, 3%~25%, 3%~24%, 3%~23%, 3%~22%, 3%~21%, 3%~20%, 3%~19%, 3%~18%, 3%~17%, 3%~16%, 3%~15%, 3%~14 %, 3%~13%, 3%~12%, 3%~11%, 3%~10%, 4%~25%, 4%~24%, 4%~23%, 4%~22%, 4%~21%, 4%~20%, 4%~19%, 4%~18%, 4%~17%, 4%~16%, 4%~15%, 4%~14%, 4%~13%, 4%~1 2%, 4%~11%, 4%~10%, 5%~25%, 5%~24%, 5%~23%, 5%~22%, 5%~21%, 5%~20%, 5%~19%, 5%~18%, 5%~17%, 5%~16%, 5%~15%, 5%~14%, 5%~13%, 5%~12%, 5%~11%, 5%~ 10%, 6%~25%, 6%~24%, 6%~23%, 6%~22%, 6%~21%, 6%~20%, 6%~19%, 6%~18%, 6%~17%, 6%~16%, 6%~15%, 6%~14%, 6%~13%, 6%~12%, 6%~11%, 6%~10%, 7%~25%, 7% ~24%, 7%~23%, 7%~22%, 7%~21%, 7%~20%, 7%~19%, 7%~18%, 7%~17%, 7%~16%, 7%~15%, 7%~14%, 7%~13%, 7%~12%, 7%~11%, 7%~10%, 8%~25%, 8%~24%, 8%~23%, 8 %~22%, 8%~21%, 8%~20%, 8%~19%, 8%~18%, 8%~17%, 8%~16%, 8%~15%, 8%~14%, 8%~13%, 8%~12%, 8%~11%, 8%~10%, 9%~25%, 9%~24%, 9%~23%, 9%~22%, 9%~21%,9%-20%, 9%-19%, 9%-18%, 9%-17%, 9%-16%, 9%-15%, 9%-14%, 9%-13%, 9%-12%, 9%-11%, 9%-10%, 10%-25%, 10%-24%, 10%-23%, 10%-22%, 10%-21%, 10%-20%, 10%-19%, 10%-18%, 10%-17%, 10%-16%, 10%-15%, 10%-14%, 10%-13%, 10%-12%, or 10%-11%.

[0135] In some embodiments, for measuring TAA-free 4-1BB induction rate, the final antibody concentration can be, but is not limited to, 100 nM as tested herein, or alternatively 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, or 50 nM.

[0136] In some embodiments, anti-CD3 antibodies or fragments considered to have balanced T cell activation activity have a TAA-dependent 4-1BB induction rate that is between 1% and 80%. In some embodiments, the TAA-dependent 4-1BB induction rate is less than or equal to 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 29%, 28%, 27%, 26% or 25%. In some embodiments, the TAA-dependent 4-1BB induction rate is at least 15%, 20%, 25%, 30%, 35%, 40% or 50%.

[0137] In some embodiments, the TAA-dependent 4-1BB induction rate for anti-CD3 antibodies or fragments considered to have balanced T cell activation activity is 15% to 80%, 15% to 70%, 15% to 60%, 15% to 50%, 15% to 40%, 15% to 35%, 20% to 80%, 20% to 70%, 20% to 60%, 20% to 50%, 20% to 40%, 20% to 35%, 20% to 30%, 25% to 80%, 25% to 70%, 25% to 60%, 25% to 50%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, 30% to 40%, or 30% to 35%.

[0138] In some embodiments, for measuring TAA-dependent 4-1BB induction rate, the final concentration of the antibody can be, but is not limited to, 1 nM as described above, or alternatively 0.5 nM, 2 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, 50 nM or 100 nM. In one embodiment, the concentration is 5 nM. In one embodiment, the concentration is 10 nM. In one embodiment, the concentration is 20 nM.

[0139] In some embodiments, the multispecific antibody has a non-TAA 4-1BB induction rate of 5%, 10%, 15%, 20% or 25% or less at an antibody concentration of 100 nM. In some embodiments, the multispecific antibody has a non-TAA 4-1BB induction rate of 15%, 14%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% or less at an antibody concentration of 100 nM. In some embodiments, measurements are made at alternative antibody concentrations of 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM or 200 nM.

[0140] In some embodiments, the multispecific antibody has a TAA-dependent 4-1BB induction rate of at least 10% at 1 nM. In some embodiments, the multispecific antibody has a TAA-dependent 4-1BB induction rate of at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40% at an antibody concentration of 1 nM. In some embodiments, measurements are made at surrogate antibody concentrations of 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM or 50 nM. T cell engagers that synergize CD3 and 4-1BB agonism

[0141] Anti-CD3 agonist antibodies can activate 4-1BB induction. As provided above, the 4-1BB induction activity of anti-CD3 antibodies can be made "dependent" on the presence of tumor antigen-expressing cells. That is, in the absence of tumor antigen, the 4-1BB induction rate is low, and in the presence of tumor antigen, the 4-1BB induction rate is many times higher. Such tumor antigen dependency is beneficial to patients because it reduces off-target toxicity.

[0142] In this regard, it is further revealed herein that both tumor antigen-dependent and tumor antigen-dependent 4-1BB induction can be synergistically enhanced when the T cell engager contains both anti-CD3 and anti-4-1BB units. In particular, when the anti-4-1BB unit itself is a tumor antigen-dependent agonistic antibody, the synergistic effect is further enhanced. A tumor antigen-dependent agonistic anti-4-1BB antibody does not activate (or has low activation ability) 4-1BB signaling in the absence of tumor antigen, but activates 4-1BB signaling in the presence of tumor antigen. Such an anti-4-1BB antibody is called a "tumor antigen-dependent agonistic anti-4-1BB antibody" or a "non-agonistic anti-4-1BB antibody".

[0143] Interestingly, such synergy between anti-CD3 and anti-4-1BB antibodies has not been observed between anti-CD3 and antibodies targeting other T cell costimulators, such as CD28, and is therefore entirely unexpected.

[0144] Another surprising finding is that when both anti-CD3 and anti-4-1BB units are included in a multispecific antibody, they are preferably located "away" from each other. For example, in either the 1+1 format or the 2+Lc2 format as shown in FIG. 9A, the anti-CD3 and anti-4-1BB units are preferably not located at the same end of the Fc fragment, but are separated by the Fc fragment. In contrast, when an anti-TAA unit is also included, the anti-CD3 and anti-TAA units are preferably located at the same side (e.g., N-terminus) of the Fc fragment.

[0145] The dramatic efficacy of such multispecific antibodies is demonstrated in Example 19 using an in vivo animal model of B16F10. B16F10 is a well-known PD1 non-responsive and resistant model, commonly referred to as "cold tumor". Cold tumors are by definition particularly difficult to treat. As expected, anti-PD1 treatment showed no efficacy, as shown in Figure 15. Bispecific antibodies exclusively targeting TAAs and either CD3 or 4-1BB (CTM01-01A, which does not target 4-1BB, and CTM01-01B, which does not target CD3) had suboptimal efficacy. A trispecific antibody targeting all of TAAs, CD3 and 4-1BB (CTM01-01) had much better efficacy. Thus, this example demonstrates the unexpectedly impressive efficacy of targeting both CD3 and 4-1BB in the manner disclosed herein in the treatment of cold tumors.

[0146] Surprisingly, the synergistic effect of CD3 signaling and 4-1BB signaling is even more evident when the anti-CD3 unit and the anti-4-1BB unit are constructed into one antibody, as demonstrated in Example 23. The TAA-CD3-4-1BB trispecific antibody showed greater T cell activation compared to the combination of the TAA-CD3 and TAA-4-1BB bispecific antibodies, suggesting a unique advantage of the TAA-CD3-4-1BB trispecific format.

[0147] Thus, one embodiment of the present disclosure provides a multispecific antibody comprising an anti-CD3 unit comprising an anti-CD3 antibody or antigen-binding fragment having binding specificity for human CD3 complex; and an anti-4-1BB unit comprising an anti-4-1BB antibody or antigen-binding fragment having binding specificity for human 4-1BB protein. In some embodiments, the multispecific antibody further comprises an anti-tumor associated antigen (TAA) unit comprising an anti-TAA antibody or antigen-binding fragment having binding specificity for human TAA.

[0148] In some embodiments, the multispecific antibody further comprises an Fc fragment. An example of a multispecific antibody format with an Fc fragment is shown in Figure 9A. In some embodiments, the anti-CD3 unit and the anti-TAA unit are both located at the N-terminus of the Fc fragment.

[0149] In the 1+1 format, the anti-CD3 antibody or antigen-binding fragment is a single chain fragment (scFv) fused to the N-terminus of one chain of the Fc fragment, and the anti-TAA antibody or antigen-binding fragment is a Fab fragment fused to the other chain of the Fc fragment.

[0150] In the 2+Lc2 format, the anti-TAA unit contains two anti-TAA Fabs fused via their respective heavy chains to the N-terminus of the Fc fragment, and the anti-CD3 unit contains two scFvs each fused to the C-terminus of the light chain of the Fab of the anti-TAA unit.

[0151] In both described formats, the anti-4-1BB unit may be fused to the C-terminus of the Fc fragment. In some embodiments, the anti-4-1BB unit comprises two anti-4-1BB nanobodies or scFvs.

[0152] In some embodiments, anti-4-1BB antibody or antigen-binding fragment is tumor-associated antigen-dependent agonist antibody or antigen-binding fragment.In some embodiments, anti-4-1BB antibody or antigen-binding fragment does not form 4-1BB protein cluster on cell surface.Such antibodies are known in the art, for example, 1A10 from Abl Bio and utomirumab (PF-05082566) from Pfizer.

[0153] Other examples are tested in Example 11 (Table B). In one embodiment, the anti-4-1BB unit comprises one or two nanobodies. In one embodiment, the nanobody comprises the CDR sequence (CDR1, CDR2 and CDR3) of SEQ ID NO: 80. Exemplary nanobody sequences include any one of SEQ ID NOs: 80-85, together with their biological equivalents (having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the reference nanobody and further retaining the CDR sequences of the reference antibody). In one embodiment, the nanobody comprises the CDR sequence of SEQ ID NO: 86. Exemplary nanobody sequences include any one of SEQ ID NOs: 86-91, together with their biological equivalents. In one embodiment, the nanobody comprises the CDR sequence of SEQ ID NO: 92. Exemplary nanobody sequences include any one of SEQ ID NOs: 92-96, together with their biological equivalents. In one embodiment, the nanobody comprises the CDR sequence of SEQ ID NO: 97. Examples of Nanobody sequences include any one of SEQ ID NOs: 97-101, along with their biological equivalents.

[0154] Human 4-1BB, as a member of the tumor necrosis factor receptor (TNFR) superfamily (TNFRSF), contains four cysteine-rich domains (CRDs) in the N-terminal extracellular region bound to a C-terminal cytoplasmic region containing a TNF receptor-associated factor (TRAF) binding motif to initiate subsequent signal transduction. The locations of these four CRDs, CRD1, CRD2, CRD3 and CRD4, are shown in Table A.

[0155] 4-1BB signaling activation is the expected mechanism for agonistic antibodies, such as utomilumab (PF-05082566) and urelumab (BMS-663513). Urelumab binds to CRD1, and utomilumab binds to CRD3. The preferred anti-4-1BB antibody or fragment for incorporation into the multispecific antibody of the present disclosure does not bind to CRD1. Instead, it binds to CRD2, CRD3 or CRD4, or a combination thereof. In some embodiments, such anti-4-1BB antibody or antigen-binding fragment does not have agonistic activity. [Table A-1] [Table A-2] [Table B-1] [Table B-2] [Table B-3] [Table B-4] [Table B-5] [Table B-6] [Table B-7-1] [Table B-7-2] [Table B-8]

[0156] A "tumor-associated antigen" or "tumor antigen" is an antigenic substance produced in tumor cells, i.e., it induces an immune response in the host. Tumor antigens are useful for identifying tumor cells and are potential candidates for use in cancer therapy. Normal proteins in the body are not antigenic. However, certain proteins are produced or overexpressed during tumor development and therefore appear "foreign" to the body. This may include normal proteins that are well isolated from the immune system, proteins that are normally produced in very small amounts, proteins that are normally produced only at certain developmental stages, or proteins whose structure is altered due to mutations.

[0157] Many tumor antigens are known in the art, and new tumor antigens can be easily identified by screening.Non-limiting examples of tumor antigens include claudin 18.2, 5T4, GPC3, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin.In certain embodiments, TAA is claudin 18.2. Examples of antibodies targeting claudin 18.2 are known in the art, including, for example, those disclosed in PCT Publications WO / 2020 / 200196, WO / 2021 / 058000, WO / 2020 / 147321, and WO / 2019 / 219089.

[0158] In some embodiments, the antibody or antigen-binding fragment binds to an antigen expressed on the surface of an immune cell. In some embodiments, the antibody or antigen-binding fragment binds to an antigen expressed on the surface of an immune cell. In some embodiments, the antibody or antigen-binding fragment binds to an antigen expressed on the surface of an immune cell. D40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50 , CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD6I, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66ECD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD79o, 0O79b, CD80, CD81, CD82, CD 83, CDw84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97 , CD98, CD99, CD100, CDIGI, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CDwl21b, CD1 22, CD123, CD124, CD125, CD126, CD127, CDwl28, CD129, CD130, CDwl31, CD132, CD134, CD135, CDw136, CDwl37, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD15G, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166, and CD182. Examples of anti-CD3 antibodies

[0159] The present disclosure also provides, in some embodiments, examples of anti-CD3 antibodies and fragments that may be used alone or selected for incorporation into T cell engagers of the present technology.

[0160] According to one embodiment of the disclosure, there is an antibody or antigen-binding fragment having binding specificity to the human CD3 complex, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3, and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.

[0161] In one embodiment, the antibody is 153A6B1 or a derivative thereof, or a humanized version thereof. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO:7, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:8, the VH CDR3 comprises the amino acid sequence of SEQ ID NO:9, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:10, the VL CDR2 comprises the amino acid sequence of SEQ ID NO:11, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:12.

[0162] VH CDR2(RIRYN G DTSYN S ALKS (SEQ ID NO: 8) has been observed to contain G55 and S61 residues (Kabat numbering) that are at risk for post-translational modification (PTM). Thus, in some embodiments, for 153A6B1 or a humanized or derived version, a G55A substitution and / or a S61A substitution can be introduced into the VH to prevent PTM. An alternative VH CDR2 with such changes is RIRYN. A DTSYNSALKS (SEQ ID NO:64), RIRYNGDTSYN A ALKS (SEQ ID NO: 65), or RIRYN A DTSYN A It may be ALKS (SEQ ID NO:66).

[0163] Similarly, VL CDR3(LQHG S GYT, SEQ ID NO: 12) contains an S93A residue that is at risk for PTM. Thus, in some embodiments, for 153A6B1 or a humanized or derived version, an S93A substitution can be introduced into the VL to prevent PTM. With such a change, the alternative VL CDR3 is LQHG A It may be GYT (SEQ ID NO: 67).

[0164] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-28, particularly SEQ ID NOs: 23-28. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 30-32, particularly SEQ ID NOs: 30-32. In some embodiments, the VL incorporates a S93A substitution.

[0165] In some embodiments, the VH comprises SEQ ID NO:23 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:23 and the VL comprises SEQ ID NO:31. In some embodiments, the VH comprises SEQ ID NO:23 and the VL comprises SEQ ID NO:32. In some embodiments, the VH comprises SEQ ID NO:24 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:24 and the VL comprises SEQ ID NO:31. In some embodiments, the VH comprises SEQ ID NO:24 and the VL comprises SEQ ID NO:32. In some embodiments, the VH comprises SEQ ID NO:25 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:25 and the VL comprises SEQ ID NO:31. In some embodiments, the VH comprises SEQ ID NO:25 and the VL comprises SEQ ID NO:32. In some embodiments, the VH comprises SEQ ID NO:26 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:26 and the VL comprises SEQ ID NO:31. In some embodiments, the VH comprises SEQ ID NO:26 and the VL comprises SEQ ID NO:32. In some embodiments, the VH comprises SEQ ID NO:27 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:27 and the VL comprises SEQ ID NO:31. In some embodiments, the VH comprises SEQ ID NO:27 and the VL comprises SEQ ID NO:32. In some embodiments, the VH comprises SEQ ID NO:28 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:28 and the VL comprises SEQ ID NO:31. In some embodiments, the VH comprises SEQ ID NO:28 and the VL comprises SEQ ID NO:32. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL incorporates an S93A substitution.

[0166] By comparing sequence analysis and activity data, it is recognized herein that certain back mutations play an important role in determining the activity of the antibody. The back mutations 27F, 78V, 29L and 30T in VH are believed to strongly promote the T cell activation activity of the antibody, while the back mutations 37V, 71R, 48M, 67L and 93T in VH are believed to help maintain a balance of activity (grade 3 or 4).

[0167] Thus, in one embodiment, the anti-CD3 antibody or fragment of the present disclosure comprises the CDRs of antibody 153A6B1 or a risk-avoiding version thereof (as described above) and further comprises at least one of the back mutations 37V, 71R, 48M, 67L and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 37V in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 71R in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 48M in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 67L in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 93T in the VH.

[0168] In some embodiments, the anti-CD3 antibody or fragment comprises at least 37V and 71R, 37V and 48M, 37V and 67L, or 37V and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 71R and 48M, 71R and 67L, or 71R and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 48M and 67L, or 48M and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 67L and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least three of 37V, 71R, 48M, 67L, and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least four of 37V, 71R, 48M, 67L, and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises all of 37V, 71R, 48M, 67L, and 93T in the VH.

[0169] In some embodiments, the anti-CD3 antibody or fragment does not include at least one of 27F, 78V, 29L and 30T in the VH. In some embodiments, the anti-CD3 antibody or fragment does not include at least 27F and 78V, 27F and 29L, or 27F and 30T in the VH. In some embodiments, the anti-CD3 antibody or fragment does not include 78V, 29L and 30T, 27F, 29L and 30T, 27F, 78V and 30T, or 27F, 78V and 29L in the VH. In some embodiments, the anti-CD3 antibody or fragment does not include any of 27F, 78V, 29L and 30T in the VH.

[0170] In some embodiments, an anti-CD3 antibody or fragment of the present disclosure comprises the CDRs of antibody 153A6B1 or a risk-avoiding version thereof (as described above) and further comprises in the VH at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not comprise at least one of the back mutations 27F, 78V, 29L and 30T (or two, three or more of them as described above).

[0171] In some embodiments, the anti-CD3 antibody or fragment is of potency grade 4 as defined in Table 10 (e.g., 153-2, 3, 4). In some embodiments, the VH comprises SEQ ID NO:24 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:25 and the VL comprises SEQ ID NO:30. In some embodiments, the VH comprises SEQ ID NO:26 and the VL comprises SEQ ID NO:30. In some embodiments, the VH incorporates a G55A substitution, a S61A substitution, or a combination thereof. In some embodiments, the VL incorporates a S93A substitution.

[0172] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO:24, comprises the CDRs of SEQ ID NO:24, and comprises at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not comprise at least one of the back mutations 27F, 78V, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO:30, and comprises the CDRs of SEQ ID NO:30.

[0173] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO:25, comprises the CDRs of SEQ ID NO:25, and comprises at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not comprise at least one of the back mutations 27F, 78V, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO:30, and comprises the CDRs of SEQ ID NO:30.

[0174] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO:26 and comprises the CDRs of SEQ ID NO:26 and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78V, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO:30 and comprises the CDRs of SEQ ID NO:30.

[0175] In some embodiments, the anti-CD3 antibody or fragment is of potency grade 3 as defined in Table 10 (e.g., 153-7, 13). In some embodiments, the VH comprises SEQ ID NO:23 and the VL comprises SEQ ID NO:31. In some embodiments, the VH comprises SEQ ID NO:23 and the VL comprises SEQ ID NO:32. In some embodiments, the VH incorporates a G55A substitution, a S61A substitution, or a combination thereof. In some embodiments, the VL incorporates a S93A substitution.

[0176] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO:23, comprises the CDRs of SEQ ID NO:23, and comprises at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not comprise at least one of the back mutations 27F, 78V, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO:31, and comprises the CDRs of SEQ ID NO:31.

[0177] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO:23 and comprises the CDRs of SEQ ID NO:23 and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78V, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO:32 and comprises the CDRs of SEQ ID NO:32.

[0178] In one embodiment, the antibody is 155A9B1 or a derivative thereof, or a humanized version thereof. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 14, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 15, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 16, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 17.

[0179] VH CDR2(RVRYN G DTSYN S ALKS (SEQ ID NO: 14) has been observed to contain G55 and S61 residues (Kabat numbering) that are at risk for post-translational modification (PTM). Thus, in some embodiments, for 155A9B1 or a humanized or derived version, a G55A substitution and / or a S61A substitution can be introduced into the VH to prevent PTM. An alternative VH CDR2 with such changes is RVRYN. A DTSYNSALKS (SEQ ID NO:68), RVRYNGDTSYN A ALKS (SEQ ID NO: 69), or RVRYN A DTSYN A It may be ALKS (SEQ ID NO: 70).

[0180] Similarly, VL CDR3(LQHN S GYT, SEQ ID NO: 17) contains an S93A residue that is at risk for PTM. Thus, in some embodiments, for 155A9B1 or a humanized or derived version, an S93A substitution can be introduced into the VL to prevent PTM. With such a change, an alternative VL CDR3 is LQHN A It may be GYT (SEQ ID NO: 71).

[0181] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 34-41, particularly SEQ ID NOs: 3 and 36-41, or SEQ ID NOs: 36-41 alone. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 43-46, particularly SEQ ID NOs: 44-46. In some embodiments, the VL incorporates an S93A substitution.

[0182] In some embodiments, the VH comprises SEQ ID NO:36 and the VL comprises SEQ ID NO:44. In some embodiments, the VH comprises SEQ ID NO:36 and the VL comprises SEQ ID NO:45. In some embodiments, the VH comprises SEQ ID NO:36 and the VL comprises SEQ ID NO:46. In some embodiments, the VH comprises SEQ ID NO:37 and the VL comprises SEQ ID NO:44. In some embodiments, the VH comprises SEQ ID NO:37 and the VL comprises SEQ ID NO:45. In some embodiments, the VH comprises SEQ ID NO:37 and the VL comprises SEQ ID NO:46. In some embodiments, the VH comprises SEQ ID NO:38 and the VL comprises SEQ ID NO:44. In some embodiments, the VH comprises SEQ ID NO:38 and the VL comprises SEQ ID NO:45. In some embodiments, the VH comprises SEQ ID NO:38 and the VL comprises SEQ ID NO:46. In some embodiments, the VH comprises SEQ ID NO:39 and the VL comprises SEQ ID NO:44. In some embodiments, the VH comprises SEQ ID NO:39 and the VL comprises SEQ ID NO:45. In some embodiments, the VH comprises SEQ ID NO:39 and the VL comprises SEQ ID NO:46. In some embodiments, the VH comprises SEQ ID NO:40 and the VL comprises SEQ ID NO:44. In some embodiments, the VH comprises SEQ ID NO:40 and the VL comprises SEQ ID NO:45. In some embodiments, the VH comprises SEQ ID NO:40 and the VL comprises SEQ ID NO:46. In some embodiments, the VH comprises SEQ ID NO:41 and the VL comprises SEQ ID NO:44. In some embodiments, the VH comprises SEQ ID NO:41 and the VL comprises SEQ ID NO:45. In some embodiments, the VH comprises SEQ ID NO:41 and the VL comprises SEQ ID NO:46. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL incorporates an S93A substitution.

[0183] By comparing sequence analysis and activity data, it is recognized herein that certain back mutations play an important role in determining the activity of the antibody. It is believed that back mutations 27F, 78A, 29L and 30T in VH strongly promote the T cell activation activity of the antibody, while back mutations 37V, 71R, 48M, 67L and 93T in VH help to maintain the balance of activity (grade 3 or 4). In addition, back mutations 36F and 58I in VL help to maintain the balance of activity (grade 3 or 4).

[0184] Thus, in one embodiment, the anti-CD3 antibody or fragment of the present disclosure comprises the CDRs of antibody 155A9B1 or a risk-avoiding version thereof (as described above) and further comprises at least one of the back mutations 37V, 71R, 48M, 67L and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 37V in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 71R in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 48M in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 67L in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 93T in the VH.

[0185] In some embodiments, the anti-CD3 antibody or fragment comprises at least 37V and 71R, 37V and 48M, 37V and 67L, or 37V and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 71R and 48M, 71R and 67L, or 71R and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 48M and 67L, or 48M and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least 67L and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least three of 37V, 71R, 48M, 67L, and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises at least four of 37V, 71R, 48M, 67L, and 93T in the VH. In some embodiments, the anti-CD3 antibody or fragment comprises all of 37V, 71R, 48M, 67L, and 93T in the VH.

[0186] In some embodiments, the anti-CD3 antibody or fragment does not include at least one of 27F, 78A, 29L and 30T in the VH. In some embodiments, the anti-CD3 antibody or fragment does not include at least 27F and 78A, 27F and 29L, or 27F and 30T in the VH. In some embodiments, the anti-CD3 antibody or fragment does not include 78A, 29L and 30T, 27F, 29L and 30T, 27F, 78A and 30T, or 27F, 78A and 29L in the VH. In some embodiments, the anti-CD3 antibody or fragment does not include any of 27F, 78A, 29L and 30T in the VH.

[0187] Thus, in one embodiment, an anti-CD3 antibody or fragment of the disclosure comprises the CDRs of antibody 155A9B1 or a risk-avoiding version thereof (as described above) and further comprises at least one of the back mutations 36F and / or 58I in the VL.

[0188] In some embodiments, an anti-CD3 antibody or fragment of the present disclosure comprises the CDRs of antibody 155A9B1 or a risk-avoiding version thereof (as described above), and further comprises in the VH at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not comprise at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above). In some embodiments, the VL comprises at least one of the back mutations 36F and / or 58I.

[0189] In certain embodiments, the VH comprises SEQ ID NO: 37 and the VL comprises SEQ ID NO: 45. In some embodiments, the VH comprises SEQ ID NO: 37 with a G55A substitution, an S61A substitution, or a combination thereof, and the VL comprises SEQ ID NO: 45. In some embodiments, the VH comprises SEQ ID NO: 37 and the VL comprises SEQ ID NO: 45 with a S93A substitution. In some embodiments, the VH comprises SEQ ID NO: 37 with a G55A substitution, an S61A substitution, or a combination thereof, and the VL comprises SEQ ID NO: 45 with a S93A substitution.

[0190] In some embodiments, the anti-CD3 antibody or fragment is of potency grade 4 as defined in Table 10 (e.g., 155-8, 9, 10, 14, 15, 16). In some embodiments, the VH comprises SEQ ID NO: 37 and the VL comprises SEQ ID NO: 45. In some embodiments, the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 45. In some embodiments, the VH comprises SEQ ID NO: 39 and the VL comprises SEQ ID NO: 45. In some embodiments, the VH comprises SEQ ID NO: 37 and the VL comprises SEQ ID NO: 46. In some embodiments, the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 46. In some embodiments, the VH comprises SEQ ID NO: 39 and the VL comprises SEQ ID NO: 46. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL incorporates an S93A substitution.

[0191] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 37, comprises the CDRs of SEQ ID NO: 37, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 45, comprises the CDRs of SEQ ID NO: 45, and includes at least the back mutations 36F and / or 58I.

[0192] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 38, comprises the CDRs of SEQ ID NO: 38, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 45, comprises the CDRs of SEQ ID NO: 45, and includes at least the back mutations 36F and / or 58I.

[0193] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 39, comprises the CDRs of SEQ ID NO: 39, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 45, comprises the CDRs of SEQ ID NO: 45, and includes at least the back mutations 36F and / or 58I.

[0194] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 37, comprises the CDRs of SEQ ID NO: 37, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 46, comprises the CDRs of SEQ ID NO: 46, and includes at least the back mutations 36F and / or 58I.

[0195] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 38, comprises the CDRs of SEQ ID NO: 38, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 46, comprises the CDRs of SEQ ID NO: 46, and includes at least the back mutations 36F and / or 58I.

[0196] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 39, comprises the CDRs of SEQ ID NO: 39, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 46, comprises the CDRs of SEQ ID NO: 46, and includes at least the back mutations 36F and / or 58I.

[0197] In some embodiments, the anti-CD3 antibody or fragment is of potency grade 3 as defined in Table 10 (e.g., 155-2, 3, 4). In some embodiments, the VH comprises SEQ ID NO: 37 and the VL comprises SEQ ID NO: 44. In some embodiments, the VH comprises SEQ ID NO: 38 and the VL comprises SEQ ID NO: 44. In some embodiments, the VH comprises SEQ ID NO: 39 and the VL comprises SEQ ID NO: 44. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL incorporates a S93A substitution.

[0198] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 37, comprises the CDRs of SEQ ID NO: 37, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 44, comprises the CDRs of SEQ ID NO: 44, and includes at least the back mutations 36F and / or 58I.

[0199] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 38, comprises the CDRs of SEQ ID NO: 38, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 44, comprises the CDRs of SEQ ID NO: 44, and includes at least the back mutations 36F and / or 58I.

[0200] In some embodiments, the VH has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 39, comprises the CDRs of SEQ ID NO: 39, and includes at least one of the back mutations 37V, 71R, 48M, 67L and 93T (or two, three, four or more of them as described above), and does not include at least one of the back mutations 27F, 78A, 29L and 30T (or two, three or more of them as described above), and the VL has at least 85%, 90% or 95% sequence identity to SEQ ID NO: 44, comprises the CDRs of SEQ ID NO: 44, and includes at least the back mutations 36F and / or 58I.

[0201] In one embodiment, the antibody is 192A7B9 or a derivative thereof, or a humanized version thereof. In some embodiments, the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 18, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 19, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 20, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 21, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 22.

[0202] VH CDR2(RMRYN G DTSYN S ALKS (SEQ ID NO: 18) has been observed to contain G55 and S61 residues (Kabat numbering) that are at risk for post-translational modification (PTM). Thus, in some embodiments, for 192A7B9 or a humanized or derived version, a G55A substitution and / or a S61A substitution can be introduced into the VH to prevent PTM. An alternative VH CDR2 with such changes is RMRYN A DTSYNSALKS (SEQ ID NO:72), RMRYNGDTSYN A ALKS (SEQ ID NO: 73), or RMRYN A DTSYN A It may be ALKS (SEQ ID NO: 74).

[0203] Similarly, VL CDR2 (IAN SLQT, SEQ ID NO: 21) contains an S53 residue that is at risk for PTM. Thus, in some embodiments, for 192A7B9 or a humanized or derived version, an S53A substitution can be introduced into the VL to prevent PTM. With such a change, an alternative VL CDR2 is A It may be LQT (SEQ ID NO: 75).

[0204] In addition, VL CDR3 (LQHN S WYT, SEQ ID NO: 22) contains an S93A residue that is at risk for PTM. Thus, in some embodiments, for 192A7B9 or a humanized or derived version, an S93A substitution can be introduced into the VL to prevent PTM. With such a change, an alternative VL CDR3 is LQHN A It may be WYT (SEQ ID NO: 76).

[0205] In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 48-55, particularly SEQ ID NOs: 5 and 50-55, or only SEQ ID NOs: 50-55. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 57-62, particularly SEQ ID NOs: 59-62. In some embodiments, the VL incorporates an S53A substitution. In some embodiments, the VL incorporates an S93A substitution.

[0206] In some embodiments, the VH comprises SEQ ID NO:50 and the VL comprises SEQ ID NO:59. In some embodiments, the VH comprises SEQ ID NO:50 and the VL comprises SEQ ID NO:60. In some embodiments, the VH comprises SEQ ID NO:50 and the VL comprises SEQ ID NO:61. In some embodiments, the VH comprises SEQ ID NO:50 and the VL comprises SEQ ID NO:62. In some embodiments, the VH comprises SEQ ID NO:51 and the VL comprises SEQ ID NO:59. In some embodiments, the VH comprises SEQ ID NO:51 and the VL comprises SEQ ID NO:60. In some embodiments, the VH comprises SEQ ID NO:51 and the VL comprises SEQ ID NO:61. In some embodiments, the VH comprises SEQ ID NO:51 and the VL comprises SEQ ID NO:62. In some embodiments, the VH comprises SEQ ID NO:52 and the VL comprises SEQ ID NO:59. In some embodiments, the VH comprises SEQ ID NO:52 and the VL comprises SEQ ID NO:60. In some embodiments, the VH comprises SEQ ID NO:52 and the VL comprises SEQ ID NO:61. In some embodiments, the VH comprises SEQ ID NO:52 and the VL comprises SEQ ID NO:62. In some embodiments, the VH comprises SEQ ID NO:53 and the VL comprises SEQ ID NO:59. In some embodiments, the VH comprises SEQ ID NO:53 and the VL comprises SEQ ID NO:60. In some embodiments, the VH comprises SEQ ID NO:53 and the VL comprises SEQ ID NO:61. In some embodiments, the VH comprises SEQ ID NO:53 and the VL comprises SEQ ID NO:62. In some embodiments, the VH comprises SEQ ID NO:54 and the VL comprises SEQ ID NO:59. In some embodiments, the VH comprises SEQ ID NO:54 and the VL comprises SEQ ID NO:60. In some embodiments, the VH comprises SEQ ID NO:54 and the VL comprises SEQ ID NO:61. In some embodiments, the VH comprises SEQ ID NO:54 and the VL comprises SEQ ID NO:62. In some embodiments, the VH comprises SEQ ID NO:55 and the VL comprises SEQ ID NO:59. In some embodiments, the VH comprises SEQ ID NO:55 and the VL comprises SEQ ID NO:60. In some embodiments, the VH comprises SEQ ID NO:55 and the VL comprises SEQ ID NO:61. In some embodiments, the VH comprises SEQ ID NO:55 and the VL comprises SEQ ID NO:62. In some embodiments, the VH incorporates a G55A substitution, an S61A substitution, or a combination thereof. In some embodiments, the VL incorporates an S53A substitution.In some embodiments, the VL incorporates a S93A substitution.

[0207] In some embodiments, the anti-CD3 antibody or fragment is of potency grade 4 as defined in Table 10 (e.g., 192-7, 13). In some embodiments, the VH comprises SEQ ID NO: 50 and the VL comprises SEQ ID NO: 60. In some embodiments, the VH comprises SEQ ID NO: 50 and the VL comprises SEQ ID NO: 61.

[0208] In some embodiments, the anti-CD3 antibody or fragment is of potency grade 3 as defined in Table 10 (e.g., 192-4). In some embodiments, the VH comprises SEQ ID NO:53 and the VL comprises SEQ ID NO:59.

[0209] Certain known anti-CD3 antibodies and fragments have also been shown to have balanced T cell activation activity and may be suitably incorporated into the T cell engagers of the present disclosure. One such anti-CD3 antigen-binding fragment is the scFv from Blinatumomab (Amgen), as demonstrated in Examples 11 and 18. The VH and VL sequences of Blinatumomab are shown in Table C. Thus, in some embodiments, the anti-CD3 unit of the T cell engager comprises the VH of SEQ ID NO: 120 and the VL of SEQ ID NO: 121. [Table C]

[0210] In some embodiments, the antibody or fragment is of class IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody or fragment has antibody-dependent cellular cytotoxicity (ADCC) capability. In some embodiments, the antibody or fragment does not have ADCC capability.

[0211] It will also be understood by those skilled in the art that the antibodies disclosed herein can be modified to differ in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a specified protein can be similar to the starting sequence, e.g., have a certain percent identity to the starting sequence, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence.

[0212] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties that are not normally associated with the antibody.Exemplary modifications are described in more detail below.For example, the antibody of the present disclosure may comprise a flexible linker sequence or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0213] The antibodies, variants, or derivatives of the present disclosure include derivatives that have been modified, i.e., modified such that the covalent attachment of any type of molecule to the antibody does not prevent the antibody from binding to the epitope. By way of example, but not by way of limitation, the antibodies can be modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, and the like. Any of a great number of chemical modifications may be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the antibodies may contain one or more non-classical amino acids.

[0214] In some embodiments, the antibody may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0215] The antibody may be conjugated or fused to a detectable label, such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, combinations of these, and other such agents known in the art.

[0216] An antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0217] Metals that emit fluorescence, e.g. 152Antibodies can also be detectably labeled using Eu, or others of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).Techniques for conjugating various moieties to antibodies are well known and are described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985);Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623- 53 (1987);Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);"Analysis, Results, And Future Developments in Antibodies See, "Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)). Examples of T cell engager formats and sequences

[0218] Certain embodiments of the present disclosure provide multispecific antibodies comprising an anti-CD3 unit comprising an anti-CD3 antibody or antigen-binding fragment having binding specificity for the human CD3 complex; and an anti-4-1BB unit comprising an anti-4-1BB antibody or antigen-binding fragment having binding specificity for human 4-1BB protein.

[0219] In some embodiments, the multispecific antibody further comprises an anti-tumor associated antigen (TAA) unit comprising an anti-TAA antibody or antigen-binding fragment having binding specificity for a human TAA. In some embodiments, the multispecific antibody further comprises an Fc fragment.

[0220] In some embodiments, the anti-CD3 unit is located at the N-terminus of the Fc fragment. In some embodiments, the anti-CD3 unit is monovalent. In some embodiments, the anti-CD3 unit has the format of an scFv, Fab or nanobody.

[0221] In some embodiments, the anti-4-1BB unit is fused to the N-terminus or C-terminus of the Fc fragment or the C-terminus of the light chain. In some embodiments, the anti-4-1BB unit comprises one or two anti-4-1BB nanobodies or scFvs.

[0222] In some embodiments, the anti-TAA unit is located at the N-terminus of the Fc fragment. In some embodiments, the anti-TAA unit is located at the C-terminus of the Fc fragment. In some embodiments, the anti-TAA unit is monovalent or bivalent. In some embodiments, the anti-TAA unit has the format of scFv, Fab or nanobody.

[0223] In some embodiments, the multispecific antibody further comprises an Fc fragment, and the anti-CD3 and anti-TAA units are located at the N-terminus of the Fc fragment. In some embodiments, the anti-4-1BB unit is fused to the C-terminus of the Fc fragment. In some embodiments, the anti-CD3 and anti-TAA units are monovalent, and the anti-4-1BB unit comprises two anti-4-1BB nanobodies or scFvs.

[0224] In some embodiments, the anti-CD3 antibody or antigen-binding fragment is a single chain fragment (scFv) or a Fab fragment or a nanobody and is fused to the N-terminus of a chain of the Fc fragment. In some embodiments, the anti-TAA antibody or antigen-binding fragment is a Fab fragment or a scFv or a nanobody and is fused to another chain of the Fc fragment. The anti-4-1BB antibody or antigen-binding fragment is a Fab fragment or a nanobody or a scFv that is fused to the C-terminus of the Fc fragment.

[0225] In some embodiments, the anti-CD3 antibody is a single chain fragment (scFv) fused to the N-terminus of a chain of the Fc fragment in a monovalent format, the anti-TAA antibody or antigen-binding fragment is a Fab fragment fused to another chain of the Fc fragment in a monovalent format, and the anti-4-1BB unit is fused to the C-terminus of the Fc and is a nanobody in a bivalent format.

[0226] Sequences of T cell engagers in the form of bispecific and trispecific antibodies are provided in Table D. [Table D-1-1] [Table D-1-2] [Table D-2-1] [Table D-2-2] [Table D-3] [Table D-4-1] [Table D-4-2] [Table D-5-1] [Table D-5-2] [Table D-6-1] [Table D-6-2] [Table D-7-1] [Table D-7-2] [Table D-8-1] [Table D-8-2] [Table D-9-1] [Table D-9-2] [Table D-10-1] [Table D-10-2] [Table D-11-1] [Table D-11-2] [Table D-12-1] [Table D-12-2] Polynucleotides encoding antibodies and methods for preparing antibodies

[0227] The present disclosure also provides an isolated polynucleotide or nucleic acid molecule encoding the antibody of the present disclosure, its variant or derivative. The polynucleotide of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotide of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptide, its variant or derivative on the same polynucleotide molecule or on separate polynucleotide molecules.

[0228] Methods for making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptide of the present disclosure are fully human. Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen administration but has an inactive endogenous locus. Exemplary techniques that can be used to make such antibodies are described in U.S. Patent Nos. 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entirety.

[0229] In certain embodiments, the prepared antibodies will not provoke an adverse immune response in the animal to be treated, e.g., in humans. In one embodiment, the antigen-binding polypeptides of the present disclosure, variants or derivatives thereof are modified to reduce their immunogenicity using techniques recognized in the art. For example, the antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from non-human antibodies, typically mouse or primate antibodies, that retain or substantially retain the antigen-binding properties of the parent antibody, but are less immunogenic in humans. This can be obtained by a variety of methods, including (a) grafting the entire non-human variable domain onto a human constant region to generate a chimeric antibody; (b) grafting at least a portion of one or more of the non-human complementarity determining regions (CDRs) onto human framework and constant regions, with or without the retention of critical framework residues; or (c) grafting the entire non-human variable domain, but "cloaking" them with human-like sections by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), as well as U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762 and 6,190,370, all of which references are hereby incorporated by reference in their entireties.

[0230] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes alterations of an antibody to modify T cell epitopes (see, e.g., International Application Publication Nos. WO / 9852976 A1 and WO / 0034317 A2). For example, variable heavy and variable light chain sequences from a starting antibody are analyzed and a human T cell epitope "map" is created from each V region that indicates the location of the epitope in relation to the complementarity determining regions (CDRs) and other critical residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions that have a low risk of altering the activity of the final antibody. A variety of alternative variable heavy and variable light chain sequences, including combinations of amino acid substitutions, are designed and these sequences are then incorporated into various binding polypeptides. Typically, between 12-24 variant antibodies are generated and tested for binding and / or function. The complete heavy and light chain genes containing the modified variable and human constant regions are then cloned into expression vectors and the subsequent plasmids are introduced into cell lines for production of whole antibodies. The antibodies are then compared in appropriate biochemical and biological assays to identify optimal variants.

[0231] The binding specificity of an antigen-binding polypeptide of the disclosure can be determined by in vitro assays, such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Treatment of tumors, especially cold tumors

[0232] As described herein, the antibodies, variants or derivatives of the disclosure can be used in certain treatment and diagnostic methods.

[0233] The present disclosure further relates to antibody-based therapies, including administering the antibodies of the present disclosure to patients, such as animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including variants and derivatives thereof as described herein), and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0234] The antibodies of the present disclosure can also be used to treat or inhibit cancer. In some embodiments, a tumor antigen (e.g., claudin 18.2) is overexpressed in tumor cells. Thus, in some embodiments, a method for treating cancer in a patient in need thereof is provided. The method, in one embodiment, entails administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient expresses, overexpresses, or is induced to express the tumor antigen. Induction of gene expression can be achieved, for example, by administration of a tumor vaccine or radiation therapy.

[0235] Tumors that may be suitably treated include those of the bladder, non-small cell lung, renal, breast, urethral, ​​colon, head and neck, squamous cell, Merkel cell, gastrointestinal, gastric, esophageal, ovarian, renal, and small cell lung cancers. Thus, the antibodies of the present disclosure may be used to treat any one or more of such cancers.

[0236] In some embodiments, the tumor to be treated is particularly difficult to treat by conventional cancer immunotherapy, for example, by antibodies targeting immune checkpoints (ICPs). Such tumors may also be called "cold tumors" or "non-immunogenic tumors". As explained above, the multispecific antibodies of the present disclosure have shown dramatic efficacy in an in vivo animal model of "cold tumor" B16F10 (see, for example, Example 19). Based thereon, in some embodiments, the present disclosure provides methods and uses for treating cold tumors with the multispecific antibodies disclosed herein.

[0237] In some embodiments, non-immunogenic tumors are those that are not infiltrated by T cells, or that have defects in T cell filtration, antigen-presenting cells (APCs) or T cell activation, or that lack T cell homing to tumor beds.All of prostate cancer, pancreatic cancer, and leukemia are non-immunogenic.The majority of breast cancers (95%), the majority of colon cancers (95%), the majority of gastric cancers (87%), the majority of head and neck cancers (84%), the majority of liver cancers (83%), the majority of esophageal cancers (86%), the majority of cervical cancers (87%), and the majority of thyroid cancers (87%) are also non-immunogenic.In addition, 83% of lung cancers, 79% of bladder cancers, 77% of kidney cancers, 70% of uterine cancers, and 66% of melanomas are also non-immunogenic.

[0238] Identification of non-immunogenic or cold tumors can also be performed by measuring the type, density and location of immune cells within the tumor. For example, Galon and Bruni (Nature Reviews Drug Discovery volume 18, pages 197-218 (2019)) describe the Immunoscore, a standardized scoring system based on the quantification of two lymphocyte populations (CD3 and CD8), e.g., in excised tissue, for guided stratification of hot and cold tumors. Immunoscores range from Immunoscore 0 (I0, low density, e.g., absence, of both cell types in both regions) to I4 (high immune cell density in both locations). By classifying cancers according to their immune infiltration, this scoring system provides an immune-based classification of tumors, including the definition of "hot" (highly invasive, Immunoscore I4) and "cold" (non-invasive, Immunoscore I0) tumors.

[0239] In some embodiments, the tumor is resistant to treatment with immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, CTLA-4 inhibitors, or combinations thereof. In some embodiments, the cancer is prostate cancer, pancreatic cancer, or leukemia. In some embodiments, the cancer is breast cancer, colon cancer, gastric cancer, head and neck cancer, liver cancer, esophageal cancer, cervical cancer, or thyroid cancer. In some embodiments, the cancer is lung cancer, bladder cancer, kidney cancer, uterine cancer, or melanoma.

[0240] Additional diseases or conditions associated with increased cell survival that may be treated, prevented, diagnosed and / or prognosed by the disclosed antibodies or variants, or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, including sarcomas and carcinomas, such as fibrosarcomas. , myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, thyroid cancer, endometrial cancer, melanoma, prostate cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma These include, but are not limited to, myeloid leukoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0241] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex and diet, as well as the number of administrations, excretion rate, drug combinations, and the severity of the particular disease being treated.The judgment of such factors by medical practitioners is within the ordinary skill of the art.The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect.The amount used can be determined by the principles of pharmacology and pharmacokinetics well known in the art.

[0242] Methods of administration of antibodies, variants or derivatives thereof include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions 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 bioactive agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, drops or transdermal patch), bucally, or as an oral spray or nasal drops.

[0243] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0244] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be utilized, for example, by using an inhaler or nebulizer, and a formulation containing an aerosolizing agent.

[0245] It may be desirable to administer an antibody, polypeptide or composition of the disclosure locally to the area requiring treatment, which can be achieved by, for example and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with wound dressing after surgery, by injection, by catheter, by suppository, or by a deposit, said deposit being of a porous, non-porous or gelatinous material, including membranes, e.g., sialastic membranes, or fibers. Preferably, when administering proteins, including antibodies, of the disclosure, care should be taken to use materials to which the protein does not absorb. composition

[0246] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of the antibody and an acceptable carrier.

[0247] In specific embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans. Moreover, a "pharmaceutically acceptable carrier" will generally be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid of any type.

[0248] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be utilized as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates, or phosphates, if desired. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for adjusting osmotic tonicity, such as sodium chloride or dextrose, are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. The compositions can be formulated as suppositories using traditional binders and carriers, such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation must be suitable for the method of administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0249] In an embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Optionally, the composition may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, these ingredients are supplied either separately or mixed in unit dosage form, for example as a dry frozen powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by injection, it can be dispensed using an injection bottle containing sterile water or saline of pharmaceutical grade. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0250] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES

[0251] Example 1 Generation and testing of anti-human CD3 antibodies This example describes the generation of anti-human CD3 monoclonal antibodies using hybridoma technology.

[0252] Antigen: human CD3D and CD3E heterodimeric protein (Sino biological, CT026-H0323H).

[0253] Immunization: To generate monoclonal antibodies against human CD3, Wistar and SD rats were immunized with CD3D and CD3E heterodimeric proteins. After four rounds of immunization, sera from immunized rats were subjected to antibody titer evaluation by ELISA. Briefly, microtiter plates were coated with 100 μl / well of 0.5 or 1 μg / ml human CD3 protein in ELISA coating buffer overnight at 4°C, then blocked with 200 μl / well of 5% nonfat dry milk. Dilutions of sera from immunized mice were added to each well and incubated for 1-2 hours at 37°C. Plates were washed with PBS / Tween and then incubated with peroxidase AffiniPure goat anti-rat IgG for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. Rats with sufficient titers of anti-CD3 IgG were boosted with human CD3D and CD3E heterodimeric protein.

[0254] Cell fusion: Cell fusion was performed by electrofusion. The fused cells were plated in 50 96-well plates per fusion.

[0255] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parent cell. The subclones were screened using the same approach as the primary clones, and culture supernatants of positive clones were subjected to additional confirmatory screening by affinity ranking.

[0256] Hybridoma clones 153A6B1, 155A9B1 and 192A7B9 were selected for further analysis. The amino acid sequences of the variable regions of 153A6B1, 155A9B1 and 192A7B9 are provided below in Table 1, and the CDR sequences are summarized in Table 1A. [Table 1] [Table 1A-1] [Table 1A-2]

[0257] Example 2 Binding activity of chimeric antibodies to the CD3 antigen ELISA Testing To evaluate the binding activity of hybridoma clones 153A6B1, 155A9B1 and 192A7B9, the chimeric mAbs from these clones were subjected to ELISA tests.

[0258] Briefly, microtiter plates were coated with 100 μl / well of human, cynomolgus and mouse CD3 proteins at 1 μg / ml in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of 153A6B1, 155A9B1 and 192A7B9 antibodies starting at 15 μg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugated for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Table 2A, 153A6B1, 155A9B1 and 192A7B9 all bound to both human and cynomolgus CD3. All antibodies tested did not bind to mouse CD3. SP34 (Biointron, B6762) served as a benchmark control. The sequence of SP34 is provided in Table 2B. [Table 2A] [Table 2B] Cell-based conjugation

[0259] FACS was used to assess the binding activity of 153A6B1, 155A9B1 and 192A7B9 chimeric mAbs to human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs). SP34 (Biointron, B6762) and OKT3 (Biointron, B6928) served as benchmark controls.

[0260] Briefly, cynoPBMCs were first incubated with 153A6B1, 155A9B1 and 192A7B9 chimeric mAbs in 3-fold serial dilutions starting at 10 nM for 30 min at 4°C. Meanwhile, human PBMCs were incubated with 153A6B1, 155A9B1 and 192A7B9 chimeric mAbs in 4-fold serial dilutions starting at 10 nM for 30 min at 4°C. After washing with PBS, PE goat anti-human IgG Fc secondary antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 min at 4°C. Samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of PE was evaluated by a MACSQuant Analyzer 16. As shown in Figures 1A and 1B, 153A6B1, 155A9B1 and 192A7B9 bound to both human and cyno PBMCs with comparable potency, demonstrating cross-species reactivity.

[0261] Example 3 Binding affinity of CD3 chimeric antibodies Binding of 153A6B1, 155A9B1 and 192A7B9 antibodies to recombinant CD3D and E proteins (human CD3-his tag) was tested on Biacore using the capture method. 153A6B1, 155A9B1 and 192A7B9 mAbs were captured using a Protein A chip. Serial dilutions of human CD3-his tag protein were injected over the captured antibody for 3 min at a flow rate of 30 μL / min. Antigen was left to dissociate for 120-360 s. All experiments were performed on a Biacore T200. Data analysis was performed by the Biacore T200 evaluation software. The results are shown in Table 3. [Table 3]

[0262] Example 4 Functional activity of CD3 chimeric antibodies This example examined the functional activity of a chimeric CD3 antibody. Cell line-based functional characterization of CD3 chimeric antibodies

[0263] To evaluate the ability of CD3 chimeric antibodies to activate the CD3 signaling pathway, a commercially available CD3 NFAT luciferase reporter system was used. In this assay, Jurkat-CD3-NFAT was used as the reporter cell line. The Jurkat-CD3-NFAT cell line has been genetically modified to stably express CD3 and luciferase downstream of a response element (Genomeditech #C17940). Luciferase expression is induced by antibody binding to the CD3 receptor. Briefly, reporter cells were plated at 2.5 x 10 cells per well. 4 The cells were cultured in white 96-well plates at a density of 100 μg / ml. Antibodies were serially diluted 3-fold and added to white 96-well assay plates at final concentrations ranging from 10 nM to 0.0005 nM. After 6 h of incubation at 37°C, luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed with GraphPad software.

[0264] As shown in Figure 2, the 153A6B1, 155A9B1 and 192A7B9 antibodies all exhibited weak CD3 NFAT activity compared to OKT3, the most potent antibody tested. Compared to another benchmark, SP34, 192A7B9 showed comparable CD3 NFAT activity, while the other two clones showed even weaker activity.

[0265] Example 5 CD8 induced by CD3 chimeric antibody + 4-1BB expression in cells This example shows that CD8 induced by a chimeric CD3 antibody + The induction of 4-1BB in cells was examined.

[0266] To assess the ability of the CD3 chimeric antibody to induce 4-1BB expression, human PBMCs were cultured at 1 x 10 cells per well. 5 Chimeric antibodies were serially diluted 3-fold and added to the 96-well plate at final concentrations ranging from 10 nM to 0.0015 nM. After 48 h of incubation at 37°C, human PBMCs were collected for further analysis. After washing with PBS, samples were stained using standard procedures by incubation with the following antibodies: anti-human CD4-APC (Ebiosciene, 17-0048-42), anti-human CD8-BV510 (BD bioscience, 563919), anti-human CD137-PE (BD Pharmingen, 555956) for 30 min at room temperature in the dark. Samples were washed twice with FACS buffer. After centrifugation, the supernatant was discarded and human PBMCs were resuspended in 0.2 mL of FACS buffer. CD8 + CD137 in T cells + CD8 + T cell subsets were assessed by MACSQuant Analyzer 16.

[0267] As shown in Figure 3, all three chimeric antibodies, like SP34 and OKT3, inhibited CD8 + 4-1BB expression on the cells could be induced, although to a lesser extent.

[0268] Example 6 Humanization of CD3 antibody The 153A6B1 / 155A9B1 / 192A7B9 variable region genes were utilized to generate humanized mAbs. In the first step of this process, the 153A6B1 / 155A9B1 / 192A7B9 VH and VK amino acid sequences were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0269] For the light chain of 153A6B1, VK1-39(O12)-JK2 was the best-matched germline, and for the heavy chain of 153A6B1, VH4-59-JH3 was selected as the humanized backbone. Then, a humanized 153A6B1 CDR-grafted antibody was designed, with CDRL1, L2 and L3 grafted into the framework sequence of VK1-39(O12)-JK2, and CDRH1, H2 and H3 grafted into the framework sequence of VH4-59-JH3. Then, a 3D model was generated to determine the amino acids in the original mouse FR region sequence that are essential for antibody binding and conformation. Based on the 153A6B1 CDR-grafted antibody sequence, six additional humanized heavy chains and three additional light chains were created.

[0270] For the light chain of 155A9B1, VK1-39(O12)-JK2 was the best-matched germline, and for the heavy chain of 155A9B1, VH4-59-JH3 was selected as the humanized backbone. Then, a humanized 155A9B1 CDR-grafted antibody was designed, with CDRL1, L2 and L3 grafted into the framework sequence of VK1-39(O12)-JK2, and CDRH1, H2 and H3 grafted into the framework sequence of VH4-59-JH3. Then, a 3D model was generated to determine the amino acids in the original mouse FR region sequence that are essential for antibody binding and conformation. Based on the 155A9B1 CDR-grafted antibody sequence, six additional humanized heavy chains and three additional light chains were created.

[0271] For the light chain of 192A7B9, VK1-39(O12)-JK2 was the best-matched germline, and for the heavy chain of 192A7B9, VH4-59-JH3 was selected as the humanized backbone. Then, the humanized 192A7B9 CDR-grafted antibody was designed, and CDRL1, L2 and L3 were grafted into the framework sequence of VK1-39(O12)-JK2, and CDRH1, H2 and H3 were grafted into the framework sequence of VH4-59-JH3. Then, a 3D model was generated to determine the amino acids in the original mouse FR region sequence that are essential for antibody binding and conformation. Based on the 192A7B9 CDR-grafted antibody sequence, six additional humanized heavy chains and four additional light chains were created.

[0272] The human germline sequences used for CDR grafting, and the resulting humanized sequences, are listed in Tables 4-6. [Table 4-1] [Table 4-2] [Table 4A-1] [Table 4A-2] [Table 5-1] [Table 5-2] [Table 5A] [Table 6-1] [Table 6-2] [Table 6A-1] [Table 6A-2]

[0273] Example 7 Binding activity of humanized antibodies to the CD3 antigen ELISA Testing To evaluate the binding activity of the humanized antibody, the humanized CD3 antibody was subjected to an ELISA test.

[0274] Briefly, microtiter plates were coated with 100 μl / well of 1 μg / ml human CD3 protein in PBS overnight at 4°C, then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of humanized antibodies starting at 15 μg / ml were added to each well and incubated for 1 hour at 37°C. Plates were washed with PBS / Tween and then incubated with anti-human IgG (H&L) (goat) antibody-peroxidase conjugated for 30 minutes at 37°C. After washing, plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450 nm. As shown in Table 7, humanized CD3 antibodies bound to human CD3 with different binding strengths. Table 7. Binding activity of humanized antibodies [Table 7A] [Table 7B] [Table 7C] [Table 7D] [Table 7E] [Table 7F] [Table 7G] [Table 7H] [Table 7I] [Table 7J] Cell-based conjugation

[0275] The binding activity of the humanized antibodies to human PBMCs was assessed using FACS.

[0276] Briefly, huPBMC cells were first incubated with 3-fold serially diluted humanized CD3 antibodies for 30 min at 4° C. PE goat anti-human IgG Fc secondary antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 min at 4° C. Samples were washed twice with FACS buffer. The mean fluorescence intensity (MFI) of PE was assessed by MACSQuant Analyzer 16. As shown in FIG. 4, humanized CD3 antibodies bound to human PBMC with various binding affinities.

[0277] Example 8 Binding affinity of humanized antibodies Binding of humanized antibodies to recombinant CD3D and E proteins (human CD3-his tag) was tested on Biacore using the capture method. Humanized antibodies were captured using a Protein A chip. Serial dilutions of human CD3-his tag protein were injected over the captured antibody for 3 min at a flow rate of 30 μl / min. Antigen was left to dissociate for 120-360 s. All experiments were performed on a Biacore T200. Data analysis was performed using the Biacore T200 evaluation software. Results are shown in Table 8. Table 8. Affinities measured by Biacore [Table 8A] [Table 8B] [Table 8C-1] [Table 8C-2]

[0278] Example 9 CD3 NFAT activity induced by humanized antibodies This example examined the functional activity of a humanized CD3 antibody. Cell line-based functional characterization of CD3 humanized antibodies

[0279] To evaluate the ability of CD3 humanized antibodies to activate the CD3 signaling pathway, a commercially available CD3 NFAT luciferase reporter system was used. In this assay, Jurkat-CD3-NFAT was used as the reporter cell line. The Jurkat-CD3-NFAT cell line has been genetically modified to harbor CD3 downstream of the NFAT response element. Luciferase expression is induced by antibody binding to the CD3 reporter. Briefly, reporter cells were plated at 2.5 × 10 cells per well. 4 The cells were plated in white 96-well plates at a density of 10 μg / mL. Test antibodies were serially diluted 5-fold and added to white 96-well assay plates at final concentrations starting at 3 μg / mL. After 6 hours of incubation at 37° C., luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed with GraphPad software.

[0280] As shown in FIG. 5, the humanized CD3 antibodies exhibited a range of different CD3 activities.

[0281] Example 10 4-1BB expression induced by humanized antibodies This example evaluated the ability of CD3 humanized antibodies to activate 4-1BB expression.

[0282] Briefly, human PBMCs were cultured at 1 × 10 cells per well.5 The cells were cultured in 96-well plates at a density of 100 μg / ml. The tested antibodies were serially diluted 10-fold and added to the 96-well plates at final concentrations ranging from 20 nM to 0.0002 nM. After 48 h of incubation at 37°C, human PBMCs were collected for further analysis. After washing with PBS, the samples were stained using standard procedures by incubation with the following antibodies: anti-human CD4-APC (Ebiosciene, 17-0048-42), anti-human CD8-BV510 (BD bioscience, 563919), anti-human CD137(4-1BB)-PE (BD Pharmingen, 555956) for 30 min at room temperature in the dark. The samples were washed twice with FACS buffer. After centrifugation, the supernatant was discarded and the human PBMCs were resuspended in 0.2 mL of FACS buffer. CD8 + CD137 in T cells + (4-1BB + )CD8 + T cell subsets were assessed by MACSQuant Analyzer 16. Total CD8 + CD137 on T cells + (4-1BB + )CD8 + The percentage (%) of T cells was used to indicate 4-1BB induction:

number

[0283] Interestingly, those humanized antibodies that were tested with little or no response in the NFAT assay (grades 1-4) could be more clearly classified based on 4-1BB-inducing activity (Figure 6).

[0284] Example 11 CD3 NFAT activity induced by CD3 monoclonal and polyspecific antibodies To further define the CD3 potency, CD3-mediated NFAT activity was measured by using SP34 as a reference in TCR / CD3 effector cells. TCR / CD3 effector cells (NFAT, Promega Cat. No. J1601) were used. Upon anti-TCR / CD3 stimulation, receptor-mediated signaling induces luminescence (due to activation of NFAT), which can be detected by adding Bio-Glo™ reagent and quantitating with a luminometer. CD3 NFAT activity induced by CD3 monoclonal antibody

[0285] Briefly, TCR / CD3 effector cells were cultured at 2.5 × 10 cells per well. 4 The cells were cultured in white 96-well plates at a density of 100 μg / ml. CD3 humanized antibodies were serially diluted 4-fold and added to white 96-well assay plates at final concentrations starting from 100 nM. After 6 h of incubation at 37° C., luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed by GraphPad software.

[0286] As shown in Figure 6 and Table 10, the humanized CD3 antibodies showed various different CD3 activities. Among them, the maximum effect (maximum luminescence measurement, Emax) showing 50% or less of SP34 in terms of NFAT response was defined as grade 1 to 4. The maximum effect (maximum luminescence measurement, Emax) showing 50% or more of SP34 in terms of NFAT response was defined as grade 5 to 9. Table 9 shows the Emax induced by SP34 in the CD3-NFAT assay and its relative percentage. [Table 9-1] [Table 9-2]

[0287] The highest value of the SP34 group was set as 100% for each assay. The percentage of the highest value of the indicated antibodies to the highest value of SP34 was used to indicate CD3 T cell activation activity:

number

[0288] To further define CD3 activity, CD3 multispecific antibodies were generated and CD3 activity was further determined for the multispecific format in the presence of TAA positive cells. For bispecific antibodies, anti-CD3 in scFv form fused to the N-terminus of Fc and anti-claudin18.2 or GPC3 units in Fab form fused to the other N-terminus of Fc were constructed to be bispecific antibodies in 1+1 format (Figure 9A). For trispecific antibodies, anti-CD3 in scFv form fused to the N-terminus of the heavy chain, 5T4 in Fab form fused to the other N-terminus of Fc, and anti-4-1BB nanobody (see sequences in Table B) fused to the C-terminus of each heavy chain were constructed to be trispecific antibody 1+1 format (Figure 11A). SP34 monoclonal antibody served as a reference control.

[0289] Briefly, TCR / CD3 effector cells (NFAT) were cultured at 2.5 × 10 cells per well. 4 Target cell lines expressing human claudin 18.2 (CHO-K1-hCLDN18.2), 5T4 (CHO-K1-h5T4 and MCF7) or GPC3 (HepG2) were cultured in white 96-well plates at a density of 2.5 × 10 4(hence E:T ratio of 1:1). Test CD3-bispecific or trispecific antibodies were serially diluted and added to white 96-well assay plates at final concentrations starting from 100 nM. SP34 (Biointron, B6762) served as a benchmark control. After 6 h of incubation at 37° C., luminescence was obtained by adding luciferase substrate and measured by a microplate reader. Four-parameter logistic curve analysis was performed by GraphPad software.

[0290] Surprisingly, all CD3 antibodies belonging to grades 1-4 activity could be further defined and classified based on their TAA-dependent T cell activation activity measured in the above-mentioned assay. Based on their TAA-dependent activity in comparison with the benchmark SP34 monoclonal antibody, anti-CD3 antibodies with stronger activity than SP34 monoclonal antibody were defined as grades 3 and 4, while anti-CD3 antibodies with undetectable activity were defined as grades 1 and 2.

[0291] As shown in Figures 8A-B, all CD3 antibodies with grade 1 or 2 activity, such as 155-7 and 155-1, had undetectable CD3 activity in the presence of claudin 18.2, while all other antibodies with activity above grade 2 level showed stronger CD3 activity compared to SP34 monoclonal antibody. Similarly, as shown in Figure 8C, all tested CD3 antibodies with activity above grade 2 (e.g., 155-5 belongs to "grade 5-9 activity", 155-14 and 155-16 belong to "grade 4 activity") showed stronger activity than that induced by SP34 monoclonal antibody, and these CD3 antibodies were constructed in the presence of GPC3-expressing HepG2 cells using GPC3-CD3 bispecific antibody. Again, consistently, all tested CD3 antibodies with activity above grade 2 (e.g., 155-8 and 155-16 belong to "grade 4 activity" and 153-7 belongs to "grade 3 activity") also induced more significant CD3 activation in 5T4-high expressing cell lines when these anti-CD3 antibodies were constructed to be 5T4 x CD3 bispecific antibodies compared to the SP34 monoclonal antibody (Figure 8D-E). Interestingly, when the anti-CD3 unit from Blinatumomab (AMGEN) ("Amb"; scFv with VH of SEQ ID NO: 120 and VL of SEQ ID NO: 121) was incorporated into the bispecific (Amb / Claudin 18.2 BiAb) format, it also showed better potency than the SP34 reference antibody (Figure 8F).

[0292] Thus, all antibodies above grade 2 showed excellent TAA-dependent CD3 activity in the TAA-CD3 bispecific antibody format. Grade 3 and 4 sequences are striking because they have little or no CD3 agonism activity in terms of NFAT response in the CD3 monoclonal antibody format, and a strong NFAT response when TAA-expressing cells are engaged in the TAA-CD3 bispecific format. And, on the other hand, the maximum effect (measured as Emax) induced by the grade 3-4 sequences is at least greater than that induced by the SP34 antibody.

[0293] Example 12 Binding activity of CD3 / claudin 18.2 bispecific antibody to human CD3 on human PBMCs The following bispecific molecules were prepared in this example: Anti-CD3 from 155A9B1-8 in scFv format fused to the N-terminus of the heavy chain and claudin 18.2 in Fab format fused to the other N-terminus were constructed into two different bispecific antibody formats A and B (Figure 9A). Format A (also called "1+1 format"), represented by bispecific antibody 155-8A, contains an anti-CD3 scFv at the N-terminus (on the right) and an anti-claudin 18.2 Fab at the other end (on the left). A knob-in-hole substitution is used in the Fc region to reduce mispairing. Format B (also called "2+Lc2 format"), represented by bispecific antibody 155-8B, contains two anti-CD3 scFv fused to the C-terminus of each of the light chain variable regions of the anti-claudin 18.2 Fab.

[0294] In addition to the two bispecific antibodies, 155-8A and 155-8B, which contain the scFv of the humanized antibody 155-8, a third bispecific antibody was also prepared: this reference bispecific antibody, Xmab, was in a 1+1 format and contained the CD3 scFv from pramotamab (Xencor).

[0295] FACS was used to evaluate the binding activity of the different formats of claudin 18.2 / CD3 bispecific antibody and the reference monospecific antibodies SP34 and 155-8 to human PBMC.

[0296] Briefly, huPBMCs were first incubated with Claudin 18.2 / CD3 bispecific antibody in 4-fold serial dilutions starting at 100 nM for 30 min at 4° C. PE goat anti-human IgG Fc secondary antibody (eBioscience™, Invitrogen) was added to each well and incubated for 30 min at 4° C. Samples were washed twice with FACS buffer. PE mean fluorescence intensity (MFI) was assessed by MACSQuant Analyzer 16.

[0297] The results are shown in Figure 9B. The binding activity of bispecific antibodies 155-8A and 155-8B to huPBMC was weaker than that of Xmab as well as the reference monospecific antibody.

[0298] (Example 13) 4-1BB expression induced by CD3 / claudin 18.2 bispecific antibody This example measured the ability of different formats of CD3 / claudin 18.2 bispecific antibodies to activate 4-1BB expression.

[0299] Briefly, human PBMCs were cultured at 1 × 10 cells per well. 5 The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) or control cells (CHO-K1) were cultured in 96-well plates at a density of 2.5 × 10 4 The cells were seeded at a density of 1000 x 1000 (hence, an E:T ratio of 4:1). FACS analysis showed that the overexpression fold of claudin 18.2 in CHO-K1-hCLDN18.2 cells was approximately 90 compared to CHO-K1 cells. Therefore, the tested bispecific antibodies were serially diluted 4-fold and added to 96-well plates at final concentrations ranging from 100 nM to 0.0004 nM. After 48 h of incubation at 37 °C, human PBMCs were collected for further analysis. After washing with PBS, the samples were stained using standard procedures by incubation for 30 min at room temperature in the dark with the following antibodies: anti-human CD4-APC (Ebiosciene, 17-0048-42), anti-human CD8-BV510 (BD bioscience, 563919), anti-human CD137-PE (BD Pharmingen, 555956). Samples were washed twice with FACS buffer. After centrifugation, the supernatant was discarded and the human PBMCs were resuspended in 0.2 mL of FACS buffer. + CD137 in T cells + (4-1BB + )CD8 + T cell subsets were assessed by MACSQuant Analyzer 16.

[0300] All CD8 + CD137 on T cells + (4-1BB + )CD8 + The percentage (%) of T cells was used to indicate 4-1BB induction rates. TAA-dependent 4-1BB induction rates were obtained when tumor-associated antigen (claudin 18.2)-expressing cells were used, and TAA-free 4-1BB induction rates were obtained in the absence of these TAA-expressing cells (using CHO-K1 cells instead).

[0301] Figure 9C shows that 155-8A (1+1 format) induced a more robust response than 155-8B (2+Lc2 format). Importantly, SP34 and XmAb also inhibited claudin 18.2. - Cells (CHO-K1) and Claudin 18.2 + While 155-8A and 155-8B showed strong 4-1BB activation activity in both cells, the activity of 155-8A and 155-8B was significantly higher than that of claudin-18.2. + The IL-18 expression was significantly more pronounced in IL-18 cells, indicating their dependence on claudin 18.2 expression.

[0302] Example 14 IL-2 secretion induced by claudin 18.2 / 4-1BB bispecific antibody is CD3 dependent This example investigated whether IL-2 secretion induced by claudin 18.2 / 4-1BB bispecific antibody is CD3 dependent.

[0303] Briefly, the experiment was divided into two groups. In the first group, plates were pre-coated with CD3 antibody (clone HIT3a) and in the second group, no CD3 antibody was used. Human PBMCs were then pre-coated at 1 × 10 cells per well. 5 The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) or control cells (CHO-K1) were cultured in 96-well plates at a density of 2.5 × 10 4(hence E:T ratio of 4:1). Claudin 18.2 / 4-1BB bispecific antibody was serially diluted 4-fold and added to the 96-well assay plate at final concentrations ranging from 100 nM to 0.0061 nM. After 48 h of incubation at 37 °C, the supernatants were collected for further analysis. IL-2 was measured by TR-FRET assay (Perkin Elmer) following the manufacturer's protocol. Lance signals were detected using an Envision. Dual emissions from 615 nM (channel 1) and 665 nM (channel 2) were obtained. A standard curve was generated by plotting the Lance counts (Ch1 / Ch2 ratio against the concentration of the standard). Data were analyzed using nonlinear regression, a four-parameter logistic equation.

[0304] As shown in FIG. 10, 4-1BB-induced IL-2 secretion was indeed dependent on CD3 activation.

[0305] Example 15 Trispecific antibody stimulated IL-2 secretion This example described a trispecific antibody that interacts with claudin 18.2, CD3 and 4-1BB to enhance both T cell activation and tumor targeting. This trispecific antibody binds to three targets: the protein claudin 18.2 on tumor cells, and the proteins CD3 and 4-1BB on T cells. The target binding domain of the antibody is shown in FIG. 11A. It contains an anti-TAA / CD3 portion in a 1+1 format with two anti-4-1BB nanobodies fused to the C-terminus of the Fc fragment.

[0306] To assess the ability of the CD3 trispecific antibody to activate T cells, IL-2 secretion was examined by LANCE (Perkin Elmer). Briefly, human PBMC cells were cultured at 1 × 10 cells per well. 5 The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) or control cells (CHO-K1) were cultured in 96-well plates at a density of 2.5 × 10 4(E:T ratio of 4:1). Test trispecific antibodies were serially diluted 4-fold and added to 96-well plates at final concentrations ranging from 100 nM to 0.098 nM. After 48 h of incubation at 37°C, supernatants were collected for further analysis. IL2 was measured by TR-FRET assay according to the manufacturer's protocol. Lance signals were detected using Envision. Dual emissions from 615 nM (channel 1) and 665 nM (channel 2) were obtained. Standard curves were generated by plotting Lance counts (Ch1 / Ch2 ratio against the concentration of standards). Data were analyzed using nonlinear regression, a four-parameter logistic equation.

[0307] In the presence of CHO-K1-hCLDN18.2 cells, IL-2 secretion in human PBMCs (Figure 11B) showed a positive correlation with CD3 NFAT activity. Grade 4 antibodies, including 155-8, 155-9 and 155-14, which have slight CD3 agonist activity, induced robust cytokine release without nonspecific activation in CHO-K1 control cells. For the grade 3 antibody (155-2), nonspecific IL-2 activation was undetectable in the absence of claudin 18.2, although its IL-2 activation was less pronounced in the presence of claudin 18.2. Thus, both grade 4 and grade 3 antibodies showed excellent safety margins.

[0308] (Example 16) Trispecific antibody-induced 4-1BB expression This example evaluated the ability of the trispecific antibody to activate 4-1BB expression.

[0309] Briefly, human PBMCs were cultured at 1 × 10 cells per well. 5 The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) or control cells (CHO-K1) were cultured in 96-well plates at a density of 2.5 × 10 4The cells were seeded at a density of 1000 x 1000 (hence an E:T ratio of 4:1). The tested antibodies were serially diluted 4-fold and added to the 96-well plate at final concentrations ranging from 100 nM to 0.098 nM. After 48 h of incubation at 37 °C, human PBMCs were collected for further analysis. After washing with PBS, the samples were stained using standard procedures by incubation for 30 min at room temperature in the dark with the following antibodies: anti-human CD4-APC (Ebiosciene, 17-0048-42), anti-human CD8-BV510 (BD bioscience, 563919), anti-human CD137-PE (BD Pharmingen, 555956). The samples were washed twice with FACS buffer. After centrifugation, the supernatant was discarded and the human PBMCs were resuspended in 0.2 mL of FACS buffer. CD8 + CD137 in T cells + (4-1BB + )CD8 + T cell subsets were assessed by MACSQuant Analyzer 16.

[0310] All CD8 + CD137 on T cells + (4-1BB + )CD8 + The percentage (%) of T cells was used to indicate 4-1BB induction rates. TAA-dependent 4-1BB induction rates were obtained when tumor-associated antigen (claudin 18.2)-expressing cells were used, and TAA-free 4-1BB induction rates were obtained in the absence of these TAA-expressing cells (using CHO-K1 cells instead).

[0311] Human PBMC CD8 +4-1BB induction in cells (Figure 12) showed a positive correlation with CD3 NFAT activity. Similar to Figure 11B, grade 4 antibodies 155-8, 155-9 and 155-14 induced strong 4-1BB induction without non-specific activation in CHO-K1 control cells. Also, with the grade 3 antibody (155-2), non-specific 4-1BB activation was undetectable in the absence of claudin 18.2, although the activation was less pronounced in the presence of claudin 18.2. Again, both grade 4 and grade 3 antibodies showed excellent safety margins.

[0312] Based on the data from CD3-NFAT activity induced by CD3 units without TAA (Figure 7A-D), TAA-dependent T cell activation by TAA-CD3 bispecific antibodies (Figure 8A-E), and TAA-dependent 4-1BB induction by CD3-4-1BB-TAA trispecific antibodies (Figure 12), CD3 activity could be divided into nine categories as shown in Table 10. Grade 3 and 4 sequences stand out because they have a well-balanced activity. Because when trispecific T cell engagers contained CD3 sequences belonging to grade 3-4 activity, these trispecific antibodies had strong 4-1BB induction and T cell activation in the presence of TAA but remained silent when TAA was absent. [Table 10]

[0313] (Example 17) IL-2 secretion and cytolytic activity induced by benchmark bispecific antibodies To compare the CD3 activity of candidate antibodies with commercially available benchmark CD3 antibodies, this example constructed our CD3 and commercially available benchmark CD3 sequences in a bispecific 1+1 format as shown in Figure 9A. Xencor-BiAb represents the CD3 sequence obtained from pramotamab (Xencor), Roche-BiAb represents the CD3 sequence obtained from mosunetuzumab (Roche), Amt-BiAb represents the CD3 sequence obtained from tarlatamab (Amgen), Amb-BiAb represents the CD3 sequence obtained from blinatumomab (Amgen), and SP34-BiAb represents the CD3 sequence obtained from SP34.

[0314] This example used IL-2 secretion and cytolytic activity as readouts for CD3 activity and thus evaluated the effect of bispecific CD3 antibodies on IL-2 production and cytolytic activity of human PBMCs co-cultured with CHO-K1-hCLDN18.2 cells. IL-2 secretion induced by CD3 bispecific antibodies

[0315] To assess the ability of the CD3 bispecific antibodies to activate T cells, IL-2 secretion was examined by LANCE (Perkin Elmer).

[0316] Briefly, human PBMCs were cultured at 1 × 10 cells per well. 5 The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) or control cells (CHO-K1) were cultured in 96-well plates at a density of 2.5 × 10 4The cells were seeded at a density of 1000 x 1000 μM. The E:T ratio is therefore 4:1. Antibodies were serially diluted 5-fold and added to 96-well plates at final concentrations ranging from 100 nM to 0.032 nM. After 48 h of incubation at 37°C, the supernatants were collected for further analysis. IL2 was measured by TR-FRET assay according to the manufacturer's protocol. Lance signals were detected using an Envision. Dual emissions from 615 nM (channel 1) and 665 nM (channel 2) were obtained. A standard curve was generated by plotting the Lance counts (Ch1 / Ch2 ratio against the concentration of the standard). Data were analyzed using nonlinear regression, a four-parameter logistic equation. Cytolytic activity induced by CD3 bispecific antibodies

[0317] To assess the ability of CD3 bispecific antibodies to activate T cells, lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells was measured using the Cytotoxicity Detection Kit. PLUS (Roche).

[0318] In brief, CD8 + Cells were isolated from human PBMCs using a CD8 isolation kit (Miltenyi Biotec Inc.). + Cells: 2 x 10 cells per well 5 The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) or control cells (CHO-K1) were cultured in 96-well plates at a density of 1 × 10 4 The cells were seeded at a density of 1000 x 1000 μg / ml. The E:T ratio was therefore 20:1. Antibodies were serially diluted 4-fold and added to a 96-well assay plate at final concentrations ranging from 10 nM to 3.81E-05 nM. After 24 h of incubation at 37°C, the supernatants were collected for further analysis. LDH was assayed according to the manufacturer's protocol. The absorbance of the samples was measured at 492 nm using an ELISA reader.

[0319] As shown in FIG. 13A, in the presence of CHO-K1-hCLDN18.2 cells the test article 155-8 bispecific antibody exhibited less cytolytic activity than the benchmark bispecific antibody, however in the presence of CHO-K1 cells the 155-8 bispecific antibody also induced less non-specific cytolytic activity in contrast to the benchmark bispecific antibody.

[0320] Furthermore, with regard to IL-2 secretion (FIG. 13B), in the presence of CHO-K1-hCLDN18.2 cells, all of the bispecific antibodies tested induced IL-2 secretion comparable to the isotype control.

[0321] (Example 18) IL-2 secretion and cytolytic activity induced by benchmark trispecific antibodies To compare the CD3 activity of our candidates and commercially available benchmark CD3 antibodies, this example constructed our CD3 and commercially available benchmark CD3 sequences to be in a trispecific 1+1 format as shown in FIG. 11A. Xencor-TriAb represents the CD3 sequence obtained from pramotamab (Xencor), Roche-TriAb represents the CD3 sequence obtained from mosunetuzumab (Roche), Amt-TriAb represents the CD3 sequence obtained from tarlatamab (Amgen), and Amb-TriAb represents the CD3 sequence obtained from blinatumomab (Amgen). We used IL-2 secretion and cytolytic activity as readouts for CD3 activity, thus evaluating the effect of trispecific CD3 antibodies on IL-2 production and cytolytic activity of human peripheral blood mononuclear cells (PBMCs) co-cultured with CHO-K1-hCLDN18.2 cells. IL-2 secretion induced by CD3 trispecific antibody

[0322] To assess the ability of the CD3 trispecific antibody to activate T cells, IL-2 secretion was examined by LANCE (Perkin Elmer).

[0323] Briefly, human PBMCs were cultured at 1 × 10 cells per well. 5The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) or control cells (CHO-K1) were cultured in 96-well plates at a density of 2.5 × 10 4 The cells were seeded at a density of 1000 x 1000 μM. The E:T ratio is therefore 4:1. Antibodies were serially diluted 5-fold and added to 96-well plates at final concentrations ranging from 100 nM to 0.032 nM. After 48 h of incubation at 37°C, the supernatants were collected for further analysis. IL2 was measured by TR-FRET assay, following the manufacturer's protocol. Lance signals were detected using Envision. Dual emissions from 615 nM (channel 1) and 665 nM (channel 2) were obtained. A standard curve was generated by plotting the Lance counts (Ch1 / Ch2 ratio against the concentration of the standard). Data were analyzed using nonlinear regression, a four-parameter logistic equation. Cytolytic activity induced by CD3 trispecific antibodies.

[0324] To assess the ability of the CD3 trispecific antibody to activate T cells, lactate dehydrogenase (LDH) activity released from the cytosol of injured cells was examined using the Cytotoxicity Detection KitPLUS (Roche).

[0325] Briefly, human PBMCs were cultured at 4 × 10 cells per well. 5 The target cell line expressing claudin 18.2 (CHO-K1-hCLDN18.2) was cultured in 96-well plates at a density of 2 × 10 4 The cells were seeded at a density of 1000 x 1000 μg / ml. Thus, the E:T ratio was 10:1. Antibodies were serially diluted 5-fold and added to the 96-well plate at final concentrations ranging from 100 nM to 0.032 nM. After 48 h of incubation at 37°C, the supernatants were collected for further analysis. LDH was assayed and subsequently examined by the manufacturer's protocol. The absorbance of the samples was measured at 492 nm using an ELISA reader.

[0326] As shown in Figure 14A, in the presence of CHO-K1-hCLDN18.2 cells, the test article 155-8 trispecific antibody showed cytolytic activity comparable to trispecific antibodies using other benchmark CD3 sequences, but in the presence of CHO-K1 cells, the 155-8 trispecific antibody induced slight off-target cytolytic activity that was significantly less than other controls except Amb-TriAb. Interestingly, Amb-TriAb, whose CD3 sequence was derived from Blinatumomab (Amgen), showed slight off-target activity but still maintained similar TAA-dependent T cell activation as the 155-8 trispecific antibody.

[0327] Furthermore, for IL-2 secretion (FIG. 14B), in the presence of CHO-K1-hCLDN18.2 cells, the test article 155-8 trispecific antibody induced robust IL-2 release from human PBMCs compared to the benchmark trispecific antibody. Meanwhile, in the presence of CHO-K1 cells, the 155-8 trispecific antibody induced less non-specific IL-2 secretion in contrast to the benchmark trispecific antibodies other than Amb-TriAb. Interestingly, Amb-TriAb, which derived the CD3 sequence from Blinatumomab (Amgen), showed minor off-target activity, but still maintained similar TAA-dependent T cell activation as the 155-8 trispecific antibody.

[0328] These results indicate that the CD3 activity of 155-8 was precise enough to initiate TAA-dependent T cell activation in a 1+1 format trispecific antibody.

[0329] (Example 19) In vivo tumor treatment with trispecific antibodies This example tested the efficacy of a trispecific antibody utilizing 155-8, anti-5T4 (naptumomab, Active Biotech), and anti-4-1BB elements in treating B16F10-h5T4 tumors.

[0330] B16F10 is a murine melanoma cell line derived from a pulmonary melanoma nodule. B16F10-h5T4 cells were injected into CD3 / 4-1BB humanized mice. Treatment consisted of 100-mm 3 It started when it reached

[0331] In addition to the 155-8-based trispecific antibody (CTM01-01), control treatments included two variants of CTM01-01: one of the variants, CTM01-01A, contained an inactivating mutation in the anti-4-1BB antibody, and the other, CTM01-01B, contained an inactivating mutation in the anti-CD3 antibody.

[0332] B16F10 tumors are a well-known PD-1 unresponsive and resistant model that is widely considered one of the most difficult tumors to treat. The results are shown in Figure 15. Mouse anti-PD-1 therapy was ineffective in this model. Treatment with CTM01-01 (a wild-type trispecific antibody targeting CD3, 4-1BB, and human 5T4 as the TAA) resulted in dramatic tumor reduction. In contrast, treatment with CTM01-01B, in which the anti-CD3 portion was inactivated, had no significant effect, and treatment with CTM01-01A, in which the anti-4-1BB portion was inactivated, had suboptimal efficacy. Thus, these results demonstrate the extraordinary in vivo efficacy of the anti-CD3 / 4-1BB / h5T4 trispecific antibody.

[0333] (Example 20) Generation of antibodies against human 5T4 Hybridoma technology was used to generate anti-human 5T4 mouse clones. Hybridoma clones 14G12, 393E9, 113H5, 159D5, 24F10, 493E10, 257F1, 353H11, 367B8, 389G2, 109H7, 286B4, 37G6, 267B5, 425G1, 449H9, 49C5, 119G5, 85B10 and 95F10 were selected for further analysis.

[0334] A competitive ELISA was performed to classify the tested 5T4 mAbs based on their binding epitopes on human 5T4. According to their competitive performance with the reference mAbs, the 5T4 mAbs were divided into four bins (bins A to D) as shown in Table 11. [Table 11]

[0335] The variable region genes were used to generate humanized mAbs. In the first step of this process, the amino acid sequences of the VH and VK of the mouse antibody were compared to available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0336] Example 21 Evaluation of 5T4-targeted trispecific T cell engagers This example developed a trispecific anti-5T4 / CD3 / 4-1BB antibody using a 1+1 format as shown in FIG. 11A, which contains two copies of an anti-4-1BB nonagonistic antibody. The VH and VL sequences of the anti-5T4 antibody naptumomab were used as a reference in the TriAb 1+1 format ("Naptu-tris"). A trispecific antibody with the sequences of 14G12 and 393E9 (bin A), 159D5 (bin B) and 286B4 (bin D) was also constructed.

[0337] Example 21A Comparison of in vitro and in vivo effects of 5T4-CD3-4-1BB bearing different 5T4-binding epitopes To evaluate the ability of 5T4-CD3-4-1BB trispecific antibody to induce IL-2 secretion among different 5T4 binding epitopes, IL-2 secretion was examined by LANCE (Perkin Elmer).

[0338] During this assay, human PBMC cells were added at 1 x 10 cells per well. 5The target cell line expressing 5T4 (CHO-K1-h5T4) or control cells (CHO-K1) were cultured in 96-well plates at a density of 2.5 × 10 4 (E:T ratio of 4:1). Test trispecific antibodies were serially diluted 5-fold and added to 96-well plates at final concentrations ranging from 100 nM to 0.032 nM. After 48 h of incubation at 37°C, supernatants were collected for further analysis. IL-2 was measured by TR-FRET assay according to the manufacturer's protocol. Lance signals were detected using Envision. Dual emissions from 615 nM (channel 1) and 665 nM (channel 2) were obtained. A standard curve was generated by plotting Lance counts (Ch1 / Ch2 ratio against the concentration of standards). Data were analyzed using nonlinear regression, a four-parameter logistic equation.

[0339] As shown in FIG. 16A, the 159D5 tris.-treated group showed the highest IL-2 secretion compared to other groups, but all of the tested trispecific antibodies with different epitopes in the 5T4 moiety significantly induced IL-2 secretion, suggesting that these trispecific antibodies can induce T cell activation.

[0340] We then tested the in vivo efficacy of these 5T4-CD3-4-1BB trispecific candidate antibodies in treating B16F10-h5T4 cell-induced tumors. B16F10-h5T4 cells were inoculated into CD3 / 4-1BB humanized mice. The mean tumor size was 100 mm 3 Once this was reached, the animals were administered 2 mpk of 5T4-CD3-4-1BB antibody at a dosing frequency of twice weekly.

[0341] The results in FIG. 16B show that treatment with 286B4 trispecific antibody (5T4 portion using 286B4 clone) and Naptu trispecific antibody (5T4 portion using 5T4 sequence from naptumomab) resulted in dramatic tumor reduction, indicating that the binding epitope of the antibody against 5T4 contributes to in vivo efficacy.

[0342] Example 21B Comparison of in vitro and in vivo effects of 5T4-CD3-4-1BB within 5T4-binding epitope D We further investigated the in vitro IL-2 secretion induced by the tested trispecific antibodies bearing the 5T4 binding epitope bin D using the same assay format and protocol as described herein.

[0343] As shown in Figure 17A, all of the tested clones with epitope bin D showed comparable potency in inducing IL-2 production. Furthermore, the in vivo efficacy results in Figure 17B showed that treatment with 286B4z3p1 trispecific antibody (5T4 part using 286B4z3p1 clone, a humanized and PTM-removed form of 286B4) and 119G5 trispecific antibody (5T4 part using 119G5 clone) at 1 mg / kg resulted in dramatic tumor reduction. 286B4z3p1 represents the third humanized version (z3) of the 286B4 chimeric antibody with one CDR region (p1) with one PTM site removed.

[0344] Example 22 Selection of anti-CD3 moieties for trispecific antibodies To evaluate the CD3 activity suitable for the 5T4-CD3-4-1BB trispecific antibody, we also compared the potency of 5T4-CD3-4-1BB trispecific antibodies with different CD3 activities using the same assay format and protocol as described in Example 20.

[0345] In the presence of MCF7 and CHO-K1-h5T4 cells, IL-2 secretion in human PBMCs (FIG. 18A) showed a positive correlation with CD3 NFAT activity (155z5>155z8>155z16). The 155z8p1B [representing the eighth humanized version (155-8) of the 155A9B1 chimeric antibody with two PTM sites removed] and 155z16p1B [representing the sixteenth humanized version (155-16) of the 155A9B1 chimeric antibody with two PTM sites removed] treatment groups exhibited weaker responses compared to the 155z8 and 155z16 treatment groups, respectively. Notably, all activities of these tested 5T4-CD3-4-1BB antibodies were dependent on the expression of 5T4, indicating the strict TAA-specific potency of these antibodies.

[0346] We also tested the in vivo efficacy of trispecific antibodies with different CD3 activities. B16F10-h5T4 cells were inoculated into CD3 / 4-1BB humanized mice. 3 Once this was reached, the animals were administered 1 mpk of 5T4-CD3-4-1BB antibody at a dosing frequency of once or twice weekly.

[0347] As shown in FIG. 18B, the 155z8 trispecific / 155z16 trispecific / 155z5 trispecific treatment group exhibited potent tumor suppression.

[0348] To determine the cellular mechanisms underlying trispecific antibody treatment, we profiled tumor infiltrating and blood immune cells using the same treatment groups in a separate study and examined changes in immune cell populations by FACS analysis after a single dose at 10 mpk. As shown in Figure 19A-E, all treatments significantly induced lymphocytic infiltration and expression of T cell subtypes at the tumor site 168 hours after the first injection. CD4 + Cells and CD8 + T cells were significantly increased, whereas NK cells remained unchanged. In addition, we found increased expression of the activation marker 4-1BB / CD25 (Fig. 19F-K) and proliferation marker Ki67 (Fig. 19L-N) in CD8+ cells in the tumor. +T cells, suggesting a proinflammatory phenotype in the tumor microenvironment. + and CD8 + A significant decrease in cell counts was observed in all treatment groups, especially in the 155z5p5 trispecific group (155z5p5 represents the fifth humanized version (155-5) of the 155A9B1 chimeric antibody with one CDR region with three PTM sites removed) (Figure 20A-E). This is likely due to the margination effect often observed with immune stimulating treatments. A single treatment at 10 mg / kg did not induce obvious activation of CD4 or CD8 cells (measured by CD25 and 4-1BB expression) in peripheral blood, except for the 155z5 trispecific antibody. Again, this indicates that trispecific antibodies with anti-CD3 units of grade 3-4 activity have negligible impact on peripheral immune activation due to strict TAA-dependent activity, whereas trispecific antibodies with anti-CD3 units above grade 4 (e.g., 155z5 (same as 155-5) belongs to grade 5-9 activity) will have some level of non-TAA-dependent activity (Figure 20F-I).

[0349] Cytokine release syndrome (CRS) is a major adverse effect of CD3-based T cell engagement therapy. To determine whether our trispecific antibody can induce CRS, the levels of cytokine release, such as IFNγ / TNFα / IL-6 / IL-2, were measured by cytometric bead array (CBA) 24 hours after the first injection in all tested groups. There was no significant increase in cytokine release in the treatment groups (Figure 21A-D), indicating a relatively well-tolerated safety profile. In summary, ex vivo analysis of tumors and blood suggested intensive T cell activation and proliferation at the tumor site without evidence of peripheral activation.

[0350] (Example 23) Comparison of trispecific antibodies and combination therapy To compare the potency of the trispecific T cell engager in inducing T cell activation with the combination of TAA-CD3 and TAA-4-1BB bispecific antibodies, 5T4-CD3-4-1BB trispecific antibody, 5T4-CD3 bispecific antibody and 5T4-4-1BB bispecific antibody were generated as shown in FIG. 22A.

[0351] Briefly, human PBMCs were cultured at 1 × 10 per well. 5 The target cell line (MCF7), which expresses relatively high levels of human 5T4, was cultured in 96-well plates at a density of 2.5 × 10 4 The cells were seeded at a density of 1000 x 1000 μM. The E:T ratio is therefore 4:1. The indicated antibodies were serially diluted and added to a 96-well plate at final concentrations ranging from 1315.789 nM to 1.316 nM. After 48 h of incubation at 37°C, the supernatants were collected for further analysis. IL-2 was measured by TR-FRET assay, following the manufacturer's protocol. Lance signals were detected using an Envision. Dual emissions from 615 nM (channel 1) and 665 nM (channel 2) were obtained. A standard curve was generated by plotting the LANCE counts (Ch1 / Ch2 ratio against the concentration of the standard). Data were analyzed using nonlinear regression, a four-parameter logistic equation.

[0352] As shown in Figure 22B, the 5T4-CD3-4-1BB trispecific showed the most significant activity in inducing IL-2 secretion compared to 5T4-CD3, 5T4-4-1BB, or the combination of 5T4-CD3 and 5T4-4-1BB, suggesting a unique advantage of the TAA-CD3-4-1BB trispecific antibody in generating synergy between the CD3 and 4-1BB pathways. * * *

[0353] The present disclosure is not limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit and scope of the present disclosure. Thus, the present disclosure encompasses modifications and variations of the present disclosure, provided that they fall within the scope of the appended claims and their equivalents.

[0354] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. an anti-CD3 unit comprising an anti-CD3 antibody or antigen-binding fragment having binding specificity for the human CD3 complex; an anti-4-1BB unit comprising an anti-4-1BB antibody or antigen-binding fragment having binding specificity for human 4-1BB protein; A multispecific antibody comprising:

2. 2. The multispecific antibody of claim 1, further comprising an anti-tumor-associated antigen (TAA) unit comprising an anti-TAA antibody or antigen-binding fragment having binding specificity for a human TAA.

3. 3. The multispecific antibody of claim 2, further comprising an Fc fragment, wherein the anti-CD3 unit and the anti-TAA unit are both located N-terminally of the Fc fragment.

4. The multispecific antibody of claim 3, wherein the anti-4-1BB unit is fused to the C-terminus of the Fc fragment.

5. The multispecific antibody of claim 4, wherein the anti-4-1BB unit comprises two anti-4-1BB nanobodies or scFvs.

6. The multispecific antibody of any one of claims 3 to 5, wherein the anti-4-1BB antibody or antigen-binding fragment is a tumor-associated antigen-dependent agonist antibody or antigen-binding fragment.

7. The multispecific antibody of any one of claims 3 to 6, wherein the anti-4-1BB antibody or antigen-binding fragment binds to cysteine-rich domain 2 (CRD2), CRD3 or CRD4 of the extracellular domain of the 4-1BB protein.

8. The multispecific antibody of any one of claims 3 to 7, wherein the anti-4-1BB antibody or antigen-binding fragment binds to CRD3 or CRD4 of the extracellular domain of the 4-1BB protein.

9. 9. The multispecific antibody of any one of claims 3 to 8, wherein the anti-CD3 antibody or antigen-binding fragment is a single chain fragment (scFv) fused to the N-terminus of one chain of the Fc fragment and the anti-TAA antibody or antigen-binding fragment is a Fab fragment, or a nanobody or scFv fused to another chain of the Fc fragment.

10. 10. The multispecific antibody of claim 1, wherein the anti-CD3 units have 50% or less of the TAA-free T cell activation activity compared to the monospecific anti-CD3 antibody SP34, wherein the TAA-free T cell activation activity represents the activity of an anti-CD3 antibody to induce activation of T cells in a sample in the absence of the anti-CD3 antibody binding to TAA-expressing cells in the sample.

11. 11. The multispecific antibody of claim 1 , wherein the anti-CD3 unit has a TAA-dependent T cell activation activity that is greater than the TAA-free T cell activation activity of the SP34 antibody, and the TAA-dependent T cell activation activity refers to the activity of the anti-CD3 antibody to induce activation of T cells in a sample in the presence of the anti-CD3 antibody further comprising an anti-TAA unit that binds to TAA-expressing cells in the sample.

12. The TAA-free or TAA-dependent T cell activation activity of an anti-CD3 antibody is The anti-CD3 antibody was treated with 2.5×10 6 cells containing a reporter gene regulated by the NFAT (nuclear factor of activated T cells) response element (NFAT-RE) at a concentration ranging from 1 nM to 100 nM for 6 hours. ４ incubating with Jurkat T cells; and measuring the activity of the reporter gene; The multispecific antibody of claim 10 or 11, wherein the antibody is measured by

13. In the measurement of the TAA-dependent T cell activation activity, the Jurkat T cells were 2.5×10 ４ 13. The multispecific antibody of claim 12, wherein the antibody is mixed with a TAA-expressing cell.

14. at 100 nM, has a TAA-free 4-1BB induction rate of 5%, 10%, 15%, 20%, or 25% or less, and said TAA-free 4-1BB induction rate is Incubating human peripheral blood CD8+ cells with the multispecific antibody at a final concentration of 100 nM for 48 hours at 37°C in the absence of TAA-expressing cells; and The percentage of 4-1BB+ CD8+ cells relative to the total number of CD8+ cells is measured as the TAA-free 4-1BB induction rate. The multispecific antibody of claim 1 , wherein the antibody is measured by

15. has a TAA-dependent 4-1BB induction rate of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% at 1 nM; the TAA-dependent 4-1BB induction rate is Incubating a 1:1 ratio of human peripheral blood CD8+ cells and TAA-expressing cells with the multispecific antibody at a final concentration of 1 nM for 48 hours at 37°C; and The percentage of 4-1BB+ CD8+ cells relative to the total number of CD8+ cells is measured as the TAA-dependent 4-1BB induction rate. The multispecific antibody of claim 14, wherein the antibody is measured by

16. 16. The multispecific antibody of claim 3, wherein the anti-TAA unit comprises two anti-TAA Fabs fused via their respective heavy chains to the N-terminus of the Fc fragment, and the anti-CD3 unit comprises two scFvs each fused to the C-terminus of a light chain of a Fab of the anti-TAA unit.

17. A multispecific antibody comprising an anti-CD3 unit and an anti-tumor-associated antigen (TAA) unit, wherein the anti-CD3 unit has binding specificity for the human CD3 complex and has balanced CD3 agonist activity.

18. 18. The multispecific antibody of claim 17, wherein the anti-CD3 units have 50% or less of the TAA-free T cell activation activity compared to the monospecific anti-CD3 antibody SP34, wherein the TAA-free T cell activation activity represents the ability of an anti-CD3 antibody to induce activation of T cells in a sample in the absence of the anti-CD3 antibody binding to TAA-expressing cells in the sample.

19. the anti-CD3 unit has a TAA-dependent T cell activation activity greater than the TAA-free T cell activation activity of the SP34 antibody, and the TAA-dependent T cell activation activity is 19. The multispecific antibody of claim 18, wherein said anti-CD3 antibody further comprises an anti-TAA unit that binds to TAA-expressing cells in said sample, and ...

20. The TAA-free or TAA-dependent T cell activation activity of an anti-CD3 antibody is The anti-CD3 antibody was treated with 2.5×10 6 cells containing a reporter gene regulated by the NFAT (nuclear factor of activated T cells) response element (NFAT-RE) at a concentration ranging from 1 nM to 100 nM for 6 hours. ４ incubating with Jurkat T cells; and measuring the activity of the reporter gene; 20. The multispecific antibody of claim 18 or 19, wherein the antibody is measured by:

21. In the measurement of the TAA-dependent T cell activation activity, the Jurkat T cells were 2.5×10 ４ 21. The multispecific antibody of claim 20, wherein the antibody is mixed with a TAA-expressing cell.

22. 22. The multispecific antibody of any one of claims 17 to 21, wherein the anti-CD3 unit comprises a single chain fragment (scFv) specific for the CD3 complex.

23. 23. The multispecific antibody of any one of claims 17 to 22, wherein the anti-TAA unit comprises a Fab fragment, a nanobody, or an scFv, preferably wherein the anti-TAA unit comprises a Fab fragment or an scFv comprising a pair of a heavy chain variable region (VH) and a light chain variable region (VL).

24. The multispecific antibody according to any one of claims 17 to 23, further comprising an anti-4-1BB unit having specificity for human 4-1BB protein.

25. 25. The multispecific antibody of claim 24, wherein the anti-CD3 unit and the anti-TAA unit are both located at the N-terminus of the Fc fragment, and the anti-4-1BB unit is located at the C-terminus of the Fc fragment.

26. The multispecific antibody of claim 24 or 25, wherein the anti-4-1BB unit comprises two nanobodies specific for the human 4-1BB protein.

27. The multispecific antibody of any one of claims 24 to 26, wherein the anti-4-1BB antibody or antigen-binding fragment is a tumor-associated antigen-dependent agonist antibody or antigen-binding fragment.

28. The multispecific antibody of any one of claims 24 to 27, wherein the anti-4-1BB antibody or antigen-binding fragment binds to cysteine-rich domain 2 (CRD2), CRD3 or CRD4 of the extracellular domain of the 4-1BB protein.

29. The multispecific antibody of any one of claims 24 to 28, wherein the anti-4-1BB antibody or antigen-binding fragment binds to CRD3 or CRD4 of the extracellular domain of the 4-1BB protein.

30. 30. The multispecific antibody of any one of claims 1 to 29, wherein the TAA is selected from the group consisting of claudin 18.2, 5T4, GPC3, EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and tenascin.

31. 31. The multispecific antibody of claim 30, wherein the TAA is claudin 18.

2.

32. A multispecific antibody comprising an anti-CD3 unit and an anti-4-1BB unit, wherein the anti-CD3 unit has binding specificity for the human CD3 complex and has balanced CD3 agonist activity.

33. 33. The multispecific antibody of claim 32, wherein the anti-CD3 units have 50% or less TAA-free T cell activation activity compared to the monospecific anti-CD3 antibody SP34, wherein the TAA-free T cell activation activity represents the ability of an anti-CD3 antibody to induce activation of T cells in a sample in the absence of the anti-CD3 antibody binding to TAA-expressing cells in the sample.

34. 34. The multispecific antibody of claim 33, wherein the anti-CD3 unit has a TAA-dependent T cell activation activity that is greater than the TAA-free T cell activation activity of the SP34 antibody, and the TAA-dependent T cell activation activity refers to the activity of the anti-CD3 antibody to induce activation of T cells in a sample in the presence of the anti-CD3 antibody further comprising an anti-TAA unit that binds to TAA-expressing cells in the sample.

35. The TAA-free or TAA-dependent T cell activation activity of an anti-CD3 antibody is The anti-CD3 antibody was treated with 2.5×10 6 cells containing a reporter gene regulated by the NFAT (nuclear factor of activated T cells) response element (NFAT-RE) at a concentration ranging from 1 nM to 100 nM for 6 hours. ４ incubating with Jurkat T cells; and measuring the activity of the reporter gene; 35. The multispecific antibody of claim 33 or 34, wherein the antibody is measured by:

36. In the measurement of the TAA-dependent T cell activation activity, the Jurkat T cells were 2.5×10 ４ 36. The multispecific antibody of claim 35, wherein the antibody is mixed with a TAA-expressing cell.

37. 37. The multispecific antibody of any one of claims 32 to 36, wherein the anti-CD3 unit comprises a single chain fragment (scFv), a nanobody, or a Fab fragment that is specific for the CD3 complex, preferably wherein the anti-CD3 unit comprises an scFv.

38. 38. The multispecific antibody of any one of claims 32 to 37, wherein the anti-CD3 unit is positioned at the N-terminus of the Fc fragment and the anti-4-1BB unit is positioned at the C-terminus of the Fc fragment.

39. The multispecific antibody of any one of claims 32 to 38, wherein the anti-4-1BB unit comprises two nanobodies specific for human 4-1BB protein.

40. The multispecific antibody of any one of claims 32 to 39, wherein the anti-4-1BB antibody or antigen-binding fragment is a tumor-associated antigen-dependent agonist antibody or antigen-binding fragment.

41. The multispecific antibody of any one of claims 32 to 40, wherein the anti-4-1BB antibody or antigen-binding fragment binds to cysteine-rich domain 2 (CRD2), CRD3 or CRD4 of the extracellular domain of the 4-1BB protein.

42. The multispecific antibody of any one of claims 32 to 41, wherein the anti-4-1BB antibody or antigen-binding fragment binds to CRD3 or CRD4 of the extracellular domain of the 4-1BB protein.

43. the anti-CD3 unit comprises a heavy chain variable region (VH) comprising a VH CDR1, a VH CDR2, and a VH CDR3, and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; the VH CDR1 comprises the amino acid sequence of SEQ ID NO:7, the VH CDR2 comprises the amino acid sequence of SEQ ID NO:8, 64, 65 or 66, the VH CDR3 comprises the amino acid sequence of SEQ ID NO:9, the VL CDR1 comprises the amino acid sequence of SEQ ID NO:10, the VL CDR2 comprises the amino acid sequence of SEQ ID NO:11, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:12 or 67; the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 14, 68, 69, or 70, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 15, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 16, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 17 or 71; or the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 18, 72, 73 or 74, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 19, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 20, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 75, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or 76; 43. A multispecific antibody according to any one of claims 1 to 42.

44. 44. The multispecific antibody of claim 43, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 7, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 8, 64, 65 or 66, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 9, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 10, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 11 and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 12 or 67.

45. 45. The multispecific antibody of claim 44, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 23-28, optionally with a G55A substitution, a S61A substitution, or a combination thereof, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 and 30-32, optionally with a S93A substitution, wherein all amino acid positions are according to Kabat numbering.

46. the VH comprises the amino acid sequence of SEQ ID NO: 24 and the VL comprises the amino acid sequence of SEQ ID NO: 30; the VH comprises the amino acid sequence of SEQ ID NO:25 and the VL comprises the amino acid sequence of SEQ ID NO:30; or The VH comprises the amino acid sequence of SEQ ID NO: 26, and the VL comprises the amino acid sequence of SEQ ID NO:

30.

45. The multispecific antibody of claim 44.

47. 44. The multispecific antibody of claim 43, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 14, 68, 69 or 70, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 15, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 16, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 11 and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 17 or 71.

48. 48. The multispecific antibody of claim 47, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 36-41, optionally with a G55A substitution, a S61A substitution, or a combination thereof, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 44-46, optionally with a S93A substitution, wherein all amino acid positions are according to Kabat numbering.

49. the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 44; the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 44; the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 44; the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 45; the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 45; the VH comprises the amino acid sequence of SEQ ID NO: 39 and the VL comprises the amino acid sequence of SEQ ID NO: 45; the VH comprises the amino acid sequence of SEQ ID NO: 37 and the VL comprises the amino acid sequence of SEQ ID NO: 46; the VH comprises the amino acid sequence of SEQ ID NO: 38 and the VL comprises the amino acid sequence of SEQ ID NO: 46; or the VH comprises the amino acid sequence of SEQ ID NO: 39, and the VL comprises the amino acid sequence of SEQ ID NO: 46; 49. The multispecific antibody of claim 48.

50. 49. The multispecific antibody of claim 48, wherein the VH comprises the amino acid sequence of SEQ ID NO: 37, optionally with a G55A substitution, a S61A substitution, or a combination thereof, and the VL comprises the amino acid sequence of SEQ ID NO: 45, optionally with a S93A substitution, wherein all amino acid positions are according to Kabat numbering.

51. 51. The multispecific antibody of claim 50, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 13, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 18, 72, 73 or 74, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 19, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 20, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or 75, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or 76.

52. 52. The multispecific antibody of claim 51 , wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 50-55, optionally with a G55A substitution, a S61A substitution, or a combination thereof, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 and 59-62, optionally with a S53A substitution, a S93A substitution, or a combination thereof, wherein all amino acid positions are according to Kabat numbering.

53. the VH comprises the amino acid sequence of SEQ ID NO: 50 and the VL comprises the amino acid sequence of SEQ ID NO: 60; the VH comprises the amino acid sequence of SEQ ID NO:50 and the VL comprises the amino acid sequence of SEQ ID NO:61; or the VH comprises the amino acid sequence of SEQ ID NO: 53, and the VL comprises the amino acid sequence of SEQ ID NO: 59; 52. The multispecific antibody of claim 51.

54. the anti-4-1BB unit comprises complementarity determining region 1 (CDR1), CDR2 and CDR3; (a) the CDR1 comprises the amino acid sequence of SEQ ID NO: 102, the CDR2 comprises the amino acid sequence of SEQ ID NO: 103, 114, or 115, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 104; (b) the CDR1 comprises the amino acid sequence of SEQ ID NO: 105, the CDR2 comprises the amino acid sequence of SEQ ID NO: 106, 116, or 117, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 107; (c) the CDR1 comprises the amino acid sequence of SEQ ID NO: 108, the CDR2 comprises the amino acid sequence of SEQ ID NO: 109, 118, or 119, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 110; or (d) the CDR1 comprises the amino acid sequence of SEQ ID NO: 111, the CDR2 comprises the amino acid sequence of SEQ ID NO: 112, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 113; 54. A multispecific antibody according to any one of claims 43 to 53.

55. The multispecific antibody of claim 54, wherein the anti-4-1BB unit comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 80 to 101.

56. 56. One or more polynucleotides encoding the antibody of any one of claims 1 to 55.

57. 57. A cell comprising one or more polynucleotides of claim 56.

58. 58. A composition comprising an antibody described in any one of claims 1 to 56, one or more polynucleotides described in claim 56, or a cell described in claim 57.

59. 60. A composition for treating cancer, comprising an antibody according to any one of claims 1 to 55.

60. 60. Use of an antibody according to any one of claims 1 to 55 for the manufacture of a medicament for treating cancer.

61. 61. The composition of claim 59 or the use of claim 60, wherein the cancer is a non-immunogenic tumor.

62. 62. The composition or use of claim 61, wherein the non-immunogenic tumor is not infiltrated by T cells or is deficient in T cell infiltration or T cell activation.

63. 63. The composition or use of claim 61 or 62, wherein the cancer is resistant to treatment with an immune checkpoint inhibitor.

64. 64. The composition or use of claim 63, wherein the immune checkpoint inhibitor is a PD-L1, PD-1 or CTLA4 inhibitor.

65. 65. The composition or use of any one of claims 59 to 64, wherein the cancer is prostate cancer, pancreatic cancer, leukemia, breast cancer, colon cancer, gastric cancer, head and neck cancer, liver cancer, esophageal cancer, cervical cancer, thyroid cancer, lung cancer, bladder cancer, kidney cancer, uterine cancer, or melanoma.

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