Anti-CD3 constructs and uses thereof

Abstract

The present application provides tumor microenvironment-related acidity activated anti-CD3 constructs that bind to CD3, the nucleic acid molecules encoding an amino acid sequence of the anti-CD3, the vectors comprising the nucleic acid molecules, host cells containing the vectors, methods of preparing the anti-CD3 construct, the pharmaceutical compositions containing the anti-CD3 construct, and the methods of using the anti-CD3 construct or compositions.

Description

Docket No. 20222-20003.40 ANTI-CD3 CONSTRUCTS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional application 63 / 658,312, filed on June 10, 2024, the contents of which are incorporated by reference in their entirety for all purposes. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (202222000340SEQLIST.xml; Size: 337,519 bytes; and Date of Creation: June 3, 2025) is herein incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to acidity-selective anti-CD3 constructs, such as acidic pH-biased molecules that specifically bind to CD3 and a disease associated antigen, and the uses thereof. BACKGROUND OF THE APPLICATION

[0004] Immunotherapy represents a proven approach that has been revolutionizing the treatment landscape across a spectrum of human diseases, encompassing cell proliferative disorders, autoimmunity, allergies, graft versus host diseases, and transplant rejections. Among the pivotal immunotherapeutic modalities are multispecific molecules designed to target either two or more epitopes simultaneously. Prominent examples of such molecules are antibodies against CD3 that have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide-loaded MHC molecules. These anti-CD3 based T cell engagers (TCEs) has been achieving clinical validations for disease treatment.

[0005] Conventional anti-CD3 based TCEs are significantly constrained by their suboptimal safety profile, posing a major impediment that restricts their broader clinical utility. Key safety concerns associated with conventional TCEs include on-target off-disease-site killing of normal tissue cells and cytokine release-related toxicities. Moreover, previously described acidic pH selective anti-CD3 TCEs either lack adequate potency (which is generally required 1sf-6766980Docket No. 20222-20003.40 to achieve adequate efficacy) or lack adequate pH selectivity (which is generally required to mitigate toxicities). There are substantial unmet needs to develop innovative TCEs that are potent yet safe for uses in treating human diseases, notably cancer, fibrosis, and autoimmune diseases.

[0006] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated by reference in their entirety. BRIEF SUMMARY OF THE APPLICATION

[0007] The following summary is illustrative only and is not intended to be limiting in any way. That is, the following summary is provided to introduce highlights, benefits and advantages of the novel antigen-binding molecules and the uses thereof. Thus, the following summary is not intended to identify essential features of the claimed subject matter, nor is it intended for use in determining the scope of the claimed subject matter.

[0008] Disclosed herein are sequences of monospecific and multispecific anti-CD3 constructs that each comprise an acidic pH-selective anti-CD3 antigen-binding domain such as an anti-CD3 Fab or scFv.

[0009] Disclosed are the variable regions of the heavy chain and the light chain of acidic pH- selective anti-CD3 monoclonal (Mab) antibodies, along with their respective Cluster Determination Regions (CDRs). Additionally, the CDR consensus sequences among select anti-CD3 antibody variants are provided.

[0010] Disclosed are exemplary molecular configurations of multispecific (e.g., bispecific, or trispecific) anti-CD3 constructs (or molecules).

[0011] Presented are biochemical and cell-based binding activity assessments of representative acidic pH-biased monospecific and multispecific anti-CD3 constructs.

[0012] Disclosed are the amino acid sequences of CD3xTAA multispecific constructs, including bispecific CD3-based T cell engagers (e.g., CD3xTAA TCEs), comprising an acidic pH-biased anti-CD3 antigen-binding domain and an anti-TAA antigen-binding domain, wherein the TAA is a tumor associated antigen.

[0013] Disclosed are also the amino acid sequences of multispecific anti-CD3 constructs, including bispecific CD3-based T cell engagers that comprise an acidic pH-biased anti-CD3 2sf-6766980Docket No. 20222-20003.40 antigen-binding domain and a disease antigen binding domain that binds a tumor antigen, a fibrosis disease antigen, or an autoimmune condition antigen.

[0014] Presented are also biochemical and functional activity analyses of representative acidic pH-selective CD3xTAA TCEs. Exemplary functional characteristics include (a) the acidic pH-selective CD3 binding activity, (b) the acidic pH-selective T cell dependent cytotoxicity (TDCC) of CD3xTAA TCEs, and (c) the acidic pH-selective induction or triggering of cytokine release by these CD3xTAA TCEs. A further exemplary functional property is the functional pH-selectivity index (“FPSI”) of these CD3xTAA TCEs, wherein such FPSI value is derived from the mathematical ratio of one of the exemplified activities at about pH 7.4 versus at about pH 6.5-6.8.

[0015] Disclosed are acidic pH-selective CD3xTROP-2 multispecific molecules, i.e., CD3xTROP-2 T cell engagers, comprising an acidic pH-biased anti-CD3 antibody domain and an anti-TROP-2 antibody domain. Also provided are the biochemical and functional properties of these CD3xTROP-2 multispecific molecules, including acidic pH-selective TDCC properties, pH-selective cytokine-induction properties, and the functional pH-selectivity indices.

[0016] Disclosed are acidic pH-selective CD3xEpCAM T cell engagers, comprising a pH- sensitive anti-CD3 antibody domain and an anti-EpCAM antibody domain. Also provided are biochemical, biophysical and functional assessments of these multispecific CD3xEpCAM molecules, including their respective pH-selective TDCC properties, pH-selective cytokine induction properties, and the functional pH-selectivity index properties.

[0017] Disclosed are acidic pH-selective CD3xEGFR T cell engagers, comprising a pH- sensitive anti-CD3 antibody domain and an anti-EGFR antibody domain. Also provided are biochemical, biophysical and functional assessments of these multispecific CD3xEGFR molecules, including their respective pH-selective TDCC properties, pH-selective cytokine induction properties, and the functional pH-selectivity index properties.

[0018] Illustrated are the descriptions of exemplary uses of anti-CD3 constructs for disease treatments. Additionally illustrated are exemplary uses of select CD3xTROP-2 TCEs, demonstrating favorable profiles of efficiently suppressing tumor growth in vivo.

[0019] Disclosed are alternative therapeutic modalities or agents incorporating or expressing an acidic pH-selective anti-CD3 antibody domain described in this invention. These alternative 3sf-6766980Docket No. 20222-20003.40 modalities or agents include, but are not limited to, immunoconjugates, RNA-based therapeutics, gene therapeutics, engineered cell therapies (e.g., TCR-T cells, CAR-T cells, CAR-NK cells, NK cells, NKT cells, gene-edited T or NK cells, tissue progenitor cells, mesenchymal stem cells, stem and stem-like cells, and artificially induced cells).

[0020] Described are additional exemplary applications of pH-selective anti-CD3 constructs. Described are exemplary uses of these anti-CD3 constructs in a combination therapy with a second agent or therapy exemplified by immune checkpoint inhibitors, antibody drug conjugates, radioligand conjugates, anti-angiogenesis inhibitors, tyrosine kinase inhibitors, DNA-damage-repair or DNA-recombination inhibitors, chemotherapeutic agents, oncolytic viruses, CDK4 / CDK6 inhibitors, lactate transporter inhibitors, and vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure. The drawings may not necessarily be in scale so as to better present certain features of the illustrated subject matter. Like annotation symbols in the various drawings indicate like elements, unless otherwise stated.

[0022] Fig. 1 illustrates exemplary molecular configurations of anti-CD3 constructs which generally bear an anti “disease antigen” (e.g., tumor associated antigens, autoimmune disease target molecules) domain, an anti-CD3 antigen-binding domain, and optionally a half-life extending domain such as a human IgG Fc domain comprising an engineered fragment crystallizable (Fc) region.

[0023] Fig. 2 illustrates additional exemplary molecular formats of anti-CD3 constructs which generally comprise an anti “disease antigen” (e.g., tumor associated antigens, autoimmune disease targets) domain, an anti-CD3 antibody domain, and optionally a half-life extender domain such as an IgG Fc domain.

[0024] Fig. 3 shows alternative exemplary molecular configurations of anti-CD3 constructs comprising a single polypeptide that generally bear an anti-CD3 antigen-binding domain, one or more anti-disease-antigen domains (e.g., anti-TAA), and optionally a half-life-extending domain such as an anti-human serum albumin (HSA) single-domain antibody fragment. 4sf-6766980Docket No. 20222-20003.40

[0025] Fig. 4 is a schematic illustrating the concept that an acidic pH-selective CD3xTAA construct engages T cells in an acidity dependent manner to achieve direct tumor cell killing and induction of cytokine secretion, wherein such killing and cytokine-induction activities are much weaker or reduced in a healthy tissue microenvironment with a physiologically relevant pH.

[0026] Fig. 5 shows the enzyme-linked immunosorbent assay (ELISA) based binding of select acidic pH-biased anti-CD3 Fab variants (h10.v52 and h10.v63) and a non-pH-selective control variant (h10.v65), to the recombinant human CD3E.

[0027] Fig. 6A shows a flow cytometry-based binding activity of two acidic pH-selective anti-CD3 antibody domains (h10.v52 and h10.v63 Fabs) and a non-pH-selective control (h10.wt) towards the cell surface of human primary T cells over a pH-titration range; Fig. 6B shows the relative cell surface binding strength based on the mean fluorescence intensity data in Fig. 6A.

[0028] Fig. 7 shows the acidic pH-dependent binding to Jurkat reporter cells by two acidic pH-selective CD3xTAA constructs comprising an acidic pH-selective anti-CD3 variant (h10.v52 or h10.v63), and an anti-TROP2 binding domain (h101-1.v6b or RS7.v1).

[0029] Fig. 8 displays Gator BLI-based in vitro equilibrium-state binding to recombinant human CD3 by a few pH-selective anti-CD3 antibody variants (h10.v63, h10.v80, and h10.v84) and non-pH-selective conventional anti-CD3 clones (h10.wt and anti-CD3 (Tarla)), over a serial pH titration.

[0030] Fig. 9 shows Gator BLI-based in vitro equilibrium-state binding to recombinant human CD3 by a panel of pH-selective anti-CD3 antibody variants in comparison to a non-pH- selective anti-CD3 clone (h10.wt) over a serial pH titration.

[0031] Fig.10 shows a T cell-dependent cytotoxicity (TDCC) functional assay data for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.6, wherein an anti-TROP-2 binding domain (h101-1.v1) is used as a model anti-TAA.

[0032] Fig. 11 shows a cytokine (IL-2 and TNFα) induction functional assay data for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.6, wherein an anti-TROP-2 binding domain (h101-1.v1) was used as a model anti-TAA. 5sf-6766980Docket No. 20222-20003.40

[0033] Fig. 12 shows a cytokine (IFNγ and IL-6) induction functional assay data for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.6, wherein an anti-TROP-2 binding domain (h101-1.v1) was used as a model anti-TAA.

[0034] Fig. 13 shows a cytokine (IL-10) induction functional assay data for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.6, wherein an anti-TROP-2 binding domain (h101-1.v1) was used as a model anti-TAA.

[0035] Fig. 14 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.7, wherein an anti-EpCAM binding domain, a tandem VHH (VHH16.v2-1) is used as a model anti-TAA, and the breast cancer line (MDA-MB-468) was used a model of disease target cell.

[0036] Fig. 15 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.7, wherein the anti-EpCAM antibody (VHH16.v2-1) was used as a model anti-TAA, and the colon cancer line (HT-29) is used a model of disease target cell.

[0037] Fig. 16 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.7, wherein three different anti-TROP-2 antibodies (VHHs) used as a model anti-TAA respectively, and the ovarian cancer line (SKOV-3) was used a model of disease target cell.

[0038] Fig. 17 shows a cytokine (IFNγ and IL-2) induction functional assay result for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.7, wherein three different anti- TROP-2 antibodies (VHHs) were used as a model of anti-TAA respectively.

[0039] Fig.18 shows a cytokine (TNFα and IL-6) induction functional assay result for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.7, wherein three different anti- TROP-2 antibodies were used as a model of anti-TAA respectively.

[0040] Fig. 19 shows a cytokine (IFNγ and IL-2) induction functional assay result for a few multispecific CD3xTAA constructs under pH 7.4 versus pH 6.7, wherein three different anti- TROP-2 antibodies were used as a model of anti-TAA respectively.

[0041] Fig. 20 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a multispecific CD3xTAA construct (h10.v80xh103-1.v1) under pH 7.4 versus pH 6.6, wherein EGFR was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell. 6sf-6766980Docket No. 20222-20003.40

[0042] Fig. 21 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a pH-selective and a non-pH selective CD3xTAA constructs (h10.wt x h101-1.v1 and h10.v80 x h101-1.v1 respectively ) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0043] Fig.22 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for two pH-selective multispecific CD3xTAA constructs (h10.v84 x h101-1.v1 and h10.v154 x h101- 1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0044] Fig.23 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for two pH-selective multispecific CD3xTAA constructs (h10.v168 x h101-1.v1 and h10.v170 x h101- 1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0045] Fig.24 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for two pH-selective multispecific CD3xTAA constructs (h10.v176 x h101-1.v1 and h10.v190 x h101- 1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0046] Fig.25 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a pH-selective multispecific CD3xTAA construct (h10.v80 x h101-1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0047] Fig.26 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a non-pH selective multispecific CD3xTAA constructs (h10.wt x h101-1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0048] Fig.27 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a pH-selective multispecific CD3xTAA construct (h10.v84 x h101-1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0049] Fig.28 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a pH-selective multispecific CD3xTAA construct (h10.v154 x h101-1.v1) under pH 7.4 versus 7sf-6766980Docket No. 20222-20003.40 pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0050] Fig.29 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a pH-selective multispecific CD3xTAA construct (h10.v168 x h101-1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0051] Fig.30 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a pH-selective multispecific CD3xTAA construct (h10.v170 x h101-1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 is used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0052] Fig.31 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a pH-selective multispecific CD3xTAA construct (h10.v176 x h101-1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0053] Fig.32 shows a cytokine (IL-2, IFNγ, TNFα, IL-6)-release functional assay result for a pH-selective multispecific CD3xTAA construct (h10.v190 x h101-1.v1) under pH 7.4 versus pH 6.7, wherein TROP-2 was used as the model TAA, and the ovarian cancer line (SKOV-3) was used the model target cell.

[0054] Fig. 33 shows a cynomolgus monkey T cell-dependent cytotoxicity (TDCC) functional assay result for an exemplary pH-selective CD3xTAA (h10.v63 x h101-1.v1) and a non-pH selective control (h10.wt x h101-1.v1) under pH 7.4 versus pH 6.7, wherein anti- TROP-2 (h101-1.v1) was used as the model anti-TAA.

[0055] Fig. 34 shows a cynomolgus monkey T cell-dependent cytotoxicity (TDCC) functional assay result for an exemplary pH-selective CD3xTAA construct (h10.v80 x h101- 1.v6b) and a non-pH selective control construct (CD3(Xalu) x TROP2 (RS7.v1)) under pH 7.4 versus pH 6.7, wherein RS7.v1 (scFv format) serves as a model anti-TAA.

[0056] Fig. 35 shows the anti-tumor efficacy of an exemplary pH-selective CD3xTAA construct (h10.v63 x h101-1.v6b) in comparison to a non-pH selective control construct (h10.wt x h101-1.v1), wherein the TROP-2-positive SKOV-3 ovarian cancer line was used as a xenograft tumor model in immuno-deficient NSG mice that are engrafted with human PBMCs. 8sf-6766980Docket No. 20222-20003.40

[0057] Fig. 36 shows the anti-tumor efficacy of two exemplary pH-selective CD3xTAA constructs (h10.v52 x h101-1.v6b and h10.v63 x h101-1.v6b) in comparison to a non-pH selective control (h10.wt x h101-1.v1), wherein the TROP-2-positive MDA-MB-468, a triple negative breast cancer line was used as a xenograft tumor model in immuno-deficient NSG mice that were engrafted with human PBMCs.

[0058] Fig. 37 shows the dose-dependent anti-tumor efficacy of an exemplary pH-selective CD3xTAA construct (h10.v80 x h101-1.v6b), wherein the TROP-2-positive SKOV-3 ovarian cancer line was used as a xenograft tumor model in immuno-deficient NSG mice that were engrafted with human PBMCs.

[0059] Fig. 38 shows the dose-dependent anti-tumor efficacy of an exemplary pH-selective CD3xTAA construct (h10.v80 x h101-1.v6b), wherein the TROP-2-positive NCI-N87 gastric cancer line was used as the xenograft tumor model in immuno-deficient NSG mice that were engrafted with human PBMCs.

[0060] Fig. 39 shows the dose-dependent anti-tumor efficacy of an exemplary pH-selective CD3xTAA construct (h10.v80 x h101-1.v6b), wherein the TROP-2-positive HCC70 triple negative breast cancer line was used as a xenograft tumor model in immuno-deficient NSG mice that were engrafted with human PBMCs.

[0061] Fig. 40 shows the dose-dependent anti-tumor efficacy of an exemplary pH-selective CD3 x TAA construct (h10.v80 x h101-1.v6b) with a bivalent TROP-2 binding design, wherein the TROP-2-positive HPAF-II pancreatic cancer line was used as a xenograft tumor model in immuno-deficient NSG mice that were engrafted with human PBMCs.

[0062] Fig. 41 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a multispecific CD3 x TAA-1 construct (h10.v84 x TAA-1) with a bivalent TAA-1 binding design, under the condition of pH 7.4 versus pH 6.6, wherein the prostate cancer line (LNCAP) was used as the model target cells.

[0063] Fig. 42 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a multispecific CD3xEGFR construct (h10.v138 x h103-1.v1) with a bivalent EGFR binding design, under the condition of pH 7.4 versus pH 6.6, wherein the ovarian cancer line (SKOV- 3) was used as the model target cell.

[0064] Fig. 43 shows a T cell-dependent cytotoxicity (TDCC) functional assay result for a multispecific CD3xTAA-2 construct (h10.v138 x anti-TAA-2) with a bivalent TAA-1 binding 9sf-6766980Docket No. 20222-20003.40 design, under the condition of pH 7.4 versus pH 6.6, wherein the prostate cancer line (LNCAP) was used as the model target cells. DETAILED DESCRIPTION OF THE APPLICATION

[0065] The present application provides innovative anti-CD3 constructs (such as multispecific anti-CD3-based antibodies or multispecific CD3xTAA antibodies), humanized anti-CD3 antibody moieties, anti-CD3 monoclonal antibodies, methods of preparing the anti- CD3 constructs, methods of using the constructs (e.g., methods of treating a disease or condition, methods of modulating an immune response, or methods of modulating a cell composition).

[0066] One hallmark feature of cancer is the aerobic glycolysis metabolism first reported by Otto Warburg in the 1920s and further supported by over 10,000 scientific publications based on PubMed search results. Aerobic glycolytic metabolism coupled with disordered vasculature, poor perfusion and / or locally increased CO2 pressure, commonly leads to unique acidic tumor microenvironment characterized by a pH range of about 5.8 to about 7.0 and an average pH of about 6.6 or 6.7, in comparison to the commonly known healthy-tissue extracellular interstitial pH range of about 7.1 to about 7.5 (more commonly, about 7.4). Through complex effects on genetics, epigenetics, cell metabolism and signaling, the tumor acidity may promote cancer progression. There have been prior therapeutics research and development efforts to reverse tumor glycolytic metabolism through using H+ / lactate transporter inhibitors such as small molecule compounds for MCT1, MCT4 and CA-IX, yet little progress has been achieved in this direction (reviewed in Cancer Metastasis Rev. (2019) 38: 205-222; Annu. Rev. Phyiol. (2020) 82: 21.1-21.24).

[0067] T cell engaging antibodies (i.e., T cell engagers or TCE) are conventionally known in the arts as multispecific (e.g., bispecific or trispecific) antibodies such as those derived from the anti-CD3 clones OKT3, L2K, SP34, UCHT1, hu38E4, hu40G5c, and SK7. These conventional CD3-based TCEs are generally not selective for tumor acidic microenvironment versus normal extracellular interstitial conditions. Tumor microenvironment acidity-activated T-cell engagers can potentially improve the specificity of tumor targeting.

[0068] There is still lack of innovative anti-CD3 antibodies with adequately potent acidity- dependent target-cell killing activity (e.g., potent tumor cell killing activity under pH 6.6 or 6.7 10sf-6766980Docket No. 20222-20003.40 with near picomolar level EC50s) as well as an adequate safety profile featured by a much- reduced target-cell killing activity and a reduced cytokine induction activity under a physiologically relevant environment of around pH 7.4, in comparison to conventional TCEs.

[0069] The present application provides anti-CD3 constructs that have innovative advantageous features of: i) having an acidic pH-selective binding to CD3, wherein the anti- CD3 exhibits a medium or high affinity to CD3 under tumor-relevant acidic conditions with a pH of about 6.0 to about 6.7 (e.g., having a monovalent affinity KDranging from about 0.1 nM to about 50 nM), but has a reduced affinity to CD3 under physiologically-relevant mildly basic conditions with a pH of about 7.3 to about 7.5 (e.g., having a monovalent affinity KD ranging from about 30 nM to about 1 µM or a near undetectable level); ii) when used as a CD3xTAA T cell engager, having an acidic pH-biased functional property of inducing potent T cell- dependent cytotoxicity (TDCC) towards TAA-positive target cells under tumor-relevant acidic conditions at about pH 6.5-6.8, but having much-reduced TDCC under physiologically- relevant conditions at about pH 7.3-7.5; and iii) having a functional property of triggering a relatively low release of cytokines (such as IL-2, IFNγ, TNFα, IL-6, or IL-10) at physiologically-relevant conditions of about pH 7.3-7.5. I. Definitions

[0070] As employed herein, the term “antibody” is to be understood in its most encompassing definition. This includes, but is not limited to, monospecific antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific, trispecific, tetraspecific antibodies), full-length antibodies, antibody domains (such as Fab, Fab’, VHH, scFv), antibody heavy-chain-only structures, and any fragments thereof that retain antigen-binding capabilities. When referring to an “antibody moiety” or “antibody domain”, it denotes either a full-length antibody, a full-length IgG, or an antibody portion or fragment thereof.

[0071] A full-length antibody such as an IgG1, an IgG2 and an IgG4 comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC- 11sf-6766980Docket No. 20222-20003.40 CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (α1 heavy chain), or lgA2 (α2 heavy chain).

[0072] The term “antigen-binding fragment” or “antigen-binding domain” in one aspect refers to an antibody fragment or domain including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a scFv, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), a single chain Fc (scFc), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody such as a variable region of heavy-chain antibody (VHH) or a heavy-chain only antibody (HCAB), a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular antibody grafted to a human framework region from one or more different antibodies. In another aspect, the antigen-binding fragment also refers to a non-antibody fragment that can bind to an antigen (such as a tumor associated antigen, tumor neoantigen, or an immune-receptor target), such non-antibody fragments include, natural ligands (or the ligand’s derivative forms) of the target antigen, the extracellular domain of cognate receptors (or their derivative forms) of the target antigen, a fragment of cell surface receptor, and alternative non-antibody scaffold. In one 12sf-6766980Docket No. 20222-20003.40 example, an anti-CD40 antigen-binding fragment can be an anti-CD40 Fab, an anti-CD40 VHH, an anti-CD40 scFv, or a whole or partial fragment derived from CD40L. In another example, an anti-TGFb antigen-binding fragment can be an anti-TGFb antibody fragment, or a fragment engineered from the extracellular domain of a TGFb receptor.

[0073] The term “binding domain” can be used interchangeably with “binding moiety”, “binding module”, “binding region”, “binding arm” , “binding fragment”, or “arm”.

[0074] “Fv” is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0075] “Single-chain Fv,” also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VHand VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0076] As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants 13sf-6766980Docket No. 20222-20003.40 thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832- 3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf- Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present application and for possible inclusion in one or more claims herein. Table 1: CDR Definitions Kabat1Chothia2MacCallum3IMGT4AHo5VH CDR1 31-35 26-32 30-35 27-38 25-40 VHCDR2 50-65 53-55 47-58 56-65 58-77 VH CDR3 95-102 96-101 93-101 105-117 109-137 VL CDR1 24-34 26-32 30-36 27-38 25-40 VLCDR2 50-56 50-52 46-55 56-65 58-77 VLCDR3 89-97 91-96 89-96 105-117 109-137 1Residue numbering follows the nomenclature of Kabat et al., supra 2Residue numbering follows the nomenclature of Chothia et al., supra 3Residue numbering follows the nomenclature of MacCallum et al., supra 4Residue numbering follows the nomenclature of Lefranc et al., supra 5Residue numbering follows the nomenclature of Honegger and Plückthun, supra

[0077] The expression “variable-domain residue-numbering as in Kabat” or “amino-acid- position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or hypervariable region (HVR) of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. 14sf-6766980Docket No. 20222-20003.40

[0078] Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody.

[0079] “Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.

[0080] “Humanized” forms of non-human (e.g., rodent, camelid) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, llama, or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0081] The term “HC-CDR” and “LC-CDR” in this disclosure are used inter-changeably with “CDR-H” (or CDRH) and “CDR-L” (or CDRL), respectively. As such, HC-CDR1 is meant CDR-H1 or CDRH1; HC-CDR2 is meant CDR-H2 or CDRH2; and HC-CDR3 is meant CDR-H3 or CDRH3, so on and so forth.

[0082] A “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, 15sf-6766980Docket No. 20222-20003.40 including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B- cell hybridoma technology.

[0083] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, sulfonyl groups or other post- translational modification(s) on the antigen.

[0084] The term “multispecific” in this disclosure refers to the capability of an antigen binding molecule to bind at least two or more unique epitopes residing in a single target or in two or more distinct targets. As such, a multispecific anti-CD3 construct or an anti-CD3 antigen-binding molecule may refer to (without limitation to) a bispecific anti-CD3 T cell engager (e.g., CD3 x TAA) , a trispecific fusion molecule (e.g., CD3 x TAA1 x TAA2; CD3 x Target #1 x Target #2), or a tetra-specific molecule.

[0085] “Percent (%) of sequence identity” or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence 16sf-6766980Docket No. 20222-20003.40 identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), GAP, BESTFIT, Geneious Prime, or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program Geneious Prime or MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0086] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0087] The term “substantial identity” or “substantially identical”, when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90% or about 91%, and more preferably at least about 95% of the nucleotide bases. When applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the tool GAP, Geneious Prime or MUSCLE using default gap weights, share at least about 90% or about 91% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions or preferred amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where 17sf-6766980Docket No. 20222-20003.40 two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331 , herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (Science 256: 1443-1445, (1992)), herein incorporated by reference in its entirety. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0088] The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0089] The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“κ”) and lambda (“λ”), based on the amino acid sequences of their constant domains.

[0090] The “CH1 domain” (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0091] “Hinge region” is generally defined as a region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions. 18sf-6766980Docket No. 20222-20003.40

[0092] The “CH2 domain” of a human IgG Fc region (also referred to as “C2” domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain- domain pairing and help stabilize the CH2 domain. Burton, Mol. Immunol.22:161-206 (1985).

[0093] The “CH3 domain” (also referred to as “C3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e., from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).

[0094] The term “Fc region” or “fragment crystallizable region” herein is used to define a C- terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native- sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0095] “Fc receptor” or “FcR” describes a receptor that binds the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an 19sf-6766980Docket No. 20222-20003.40 immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daëron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126: 330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

[0096] As used herein, a first antibody or fragment thereof “competes” for binding to a target antigen with a second antibody or fragment thereof when the first antibody or fragment thereof inhibits the target antigen binding of the second antibody of fragment thereof by at least about 50% (such as at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody or fragment thereof, or vice versa. A high throughput process for “binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0097] As use herein, the terms “specifically binds,” “specifically recognizing,” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody moiety that specifically recognizes a target (which can be an epitope) is an antibody or antibody moiety that binds this target with greater affinity, avidity, more readily, and / or with greater duration than its bindings to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target has a dissociation constant (KD) of ≤ 10-5M, ≤ 10-6M, ≤ 10-7M, ≤ 10-8M, ≤ 10-9M, ≤ 10-10M, ≤ 10-11M, or ≤ 10-12M. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-tests, biolayer interferometry, surface plasma resonance, and peptide scans.

[0098] The term “tumor microenvironment” or “TME” refers to the environment around a tumor and the interstitial compositions inside the tumor, including the surrounding blood 20sf-6766980Docket No. 20222-20003.40 vessels, immune cells, fibroblasts, signaling molecules, extracellular matrix and interstitial fluid. In some embodiments, tumor microenvironment comprises the microenvironment of a tumor draining lymph node.

[0099] The term “TAA positive” and “TAA-positive” can be used interchangeably, which refers to a cell-surface expression level of a tumor associated antigen (“TAA”) at a pathologically relevant high level or at a commonly accepted high level defined in clinical or diagnostic practices. For instance, “HER2-positive” is generally defined as HER23+ based on immunohistology analysis of tumor specimen sections, or as HER2 2+ plus positive HER2 gene amplification status confirmed by FISH (fluorescent in situ hybridization). In comparison, “TAA low” or “TAA-low” refers to a cell-surface expression level of a tumor associated antigen at a low level commonly observed in normal tissues or in some tumor cells which are categorized as “TAA low” in clinical or diagnostic practices. For instance, heart cardiomyocytes are generally considered as HER2-low cells due to their low HER2 expression level. In some embodiments, a TAA positive cell means a target cell expressing a high or medium-high level of the said TAA on the said target cell’s surface. In some embodiments, a TAA expression level can be assessed by immunohistochemistry (IHC) and quantified through a so-called H-score method, which may capture both the intensity and the proportion of a TAA or a biomarker of interest from the IHC image and comprise values in a dynamic range between 0 and 300 to quantitatively reflect the TAA abundance.

[0100] The term “EC50” or “EC50” refers to the dose of a given test article or agent such as a that can reach about 50% of the maximal pharmacological activity (e.g., target-cell killing activity, the maximal growth inhibition activity, or the maximal binding activity), as compared to or normalized against about the 0% activity of a negative control (or a mock control) under a defined experimental condition. EC50 may be determined using a software tool such as GraphPad Prism, Excel, MATLAB, and R. In cases wherein the maximal pharmacological activity is yet to be reached within a given dose range, the EC50 will be considered to be more than the highest tested dose (e.g., the EC50 is “> 200nM”, if the highest tested dose is 200 nM). In cases wherein the dose-dependent pharmacological activity curve does not provide a statistically reliable EC50, the dose corresponding to about half curve-height is used as the approximate EC50. 21sf-6766980Docket No. 20222-20003.40

[0101] The term “ECmax” or “ECmax” refers to the lowest dose of a given test article or agent such as a that can reach the maximal plateau activity ( in terms of target-cell killing, or target- cell growth inhibition, or target-molecule binding). ECmax may be determined using a software tool such as GraphPad Prism, Excel, MATLAB, and R.

[0102] The term “PA30” or “PA30” generally refers to the concentration or dose of a given test article or agent that is required to achieve 30% of the maximal pharmacological activity based on a specified experimental assay condition. As such, the TDCC PA30 generally refers to the concentration of a test agent (such as a TCE) that is necessary to reach the killing of 30% target cells in a T cell dependent cytotoxicity assay. Similarly, the PA30 of a cytokine release assay for a test agent generally refers to the concentration of such test agent that is required to reach 30% of the maximally-achievable cytokine release effect in a defined cytokine release assay.

[0103] The term “functional pH-selectivity index” (or “FPSI”) in general refers to the ratio of a functional activity at a physiologically relevant pH of about 7.4 or about 7.5 over a functional activity at a tumor-relevant pH of about 6.5, 6.6, 6.7 or 6.8. For instance, an affinity- based FPSI (“affinity FPSI”) would refer to the ratio of the CD3-binding affinity at a physiologically relevant pH of about 7.4 or about 7.5 over the CD3-binding affinity at a tumor- relevant pH of about 6.5, 6.6, 6.7 or 6.8 for one and the same anti-CD3 variant or CD3xTAA construct. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.5. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.6. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.7. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.8. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.5. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.6. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.7. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.8.

[0104] Similarly, a TDCC-based FPSI (“TDCC FPSI”) herein and thereafter refers to the ratio of an EC50 (or PA30) derived from an in vitro TDCC assay at a pH of about 7.4 or about 7.5 assay condition over that derived from a pH of about 6.5, 6.6, 6.7 or 6.8 assay condition for a multispecific anti-CD3 construct or a CD3xTAA TCE. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.5. In some embodiments, the 22sf-6766980Docket No. 20222-20003.40 comparison is the said ratio at a pH of 7.4 vs. a pH of 6.6. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.7. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.8. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.5. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.6. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.7. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.8.

[0105] While the FPSI of a given test agent can be generally characterized by the foregoing affinity- or TDCC-based metrics, such a FPSI can be also calculated by alternative functional activity-based metrics such as T cell activation and cytokine induction. For example, a cytokine-induction based FPSI (“cytokine FPSI”) would refer to the ratio of an EC50 based on an in vitro cell-based cytokine release assay (such as the release of IFNγ, IL-2, IL-6, TNFα, IL- 10, and / or MCP-1) under a physiologically relevant pH (e.g., about pH 7.5 or about 7.4) versus that under a tumor relevant acidic pH (e.g., about pH 6.8, 6.7, 6.6, or 6.5) for a multispecific anti-CD3 construct or a CD3xTAA TCE molecule. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.5. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.6. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.7. In some embodiments, the comparison is the said ratio at a pH of 7.4 vs. a pH of 6.8. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.5. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.6. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.7. In some embodiments, the comparison is the said ratio at a pH of 7.5 vs. a pH of 6.8.

[0106] An “isolated” antibody or polypeptide (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment.

[0107] An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen- binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The 23sf-6766980Docket No. 20222-20003.40 isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0108] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0109] Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0110] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0111] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A 24sf-6766980Docket No. 20222-20003.40 “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0112] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0113] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of cancer (such as, for example, tumor volume). The methods of the application contemplate any one or more of these aspects of treatment.

[0114] In the context of cancer, the term “treating” includes any or all of: inhibiting growth of cancer cells, inhibiting replication of cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.

[0115] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to that of a reference. In certain embodiments, by “reduce” or 25sf-6766980Docket No. 20222-20003.40 “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, 98% or greater.

[0116] A “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy and / or non-diseased sample. In some examples, a reference may be obtained from an untreated sample. In some examples, a reference is obtained from a non-diseased or non-treated sample of an individual. In some examples, a reference is obtained from one or more healthy individuals who are not the individual or patient.

[0117] The term “benchmark” as in “benchmark clone” or “benchmark antibody” or “benchmark TCE” refers to well characterized or standard antibody clones that are used for comparison purpose. A commonly used benchmark antibody can be an antibody clone sequence that is comprised by a clinically approved antibody drug. For example, the well characterized anti-CD3 clones OKT3 and the related L2K (or a de-immunized L2K version) comprised by the FDA-approved Blinatumomab, and SP34 that is commonly adapted as a component of some conventional TCEs. In some embodiment, a benchmark is meant a reference.

[0118] As used herein, “delaying development of a disease" means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late-stage cancer, such as development of metastasis, may be delayed.

[0119] “Preventing” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.

[0120] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody which suppresses 26sf-6766980Docket No. 20222-20003.40 tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.

[0121] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0122] An “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0123] A “therapeutically effective amount” of a substance / molecule of the application, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations.

[0124] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0125] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to an individual to which the formulation would be administered. Such formulations may be sterile.

[0126] A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients 27sf-6766980Docket No. 20222-20003.40 of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0127] A “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.

[0128] Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.

[0129] The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered with a time separation of no more than about 60 minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.

[0130] The term “sequentially” is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s). For example, administration of the two or more therapeutic agents are administered with a time separation of more than about 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.

[0131] As used herein, “in conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0132] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0133] An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein. 28sf-6766980Docket No. 20222-20003.40

[0134] It is understood that embodiments of the application described herein include “consisting” and / or “consisting essentially of” embodiments.

[0135] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0136] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0137] The term “about X-Y” used herein has the same meaning as “about X to about Y.”

[0138] The term “about 7.4” used herein refers to 7.5, 7.4, 7.3 or any value between 7.3 and 7.5; similarly, the term “about 90%” refers to 91%, 90%, 89% or any value between 91% and 89%; the term “about 6.6” refers to 6.7, 6.6, 6.5 or any value between 6.7 and 6.5, so on and so forth.

[0139] The term “tissue environment” or “tumor microenvironment” refers to interstitial extracellular environment of tissue or tumor respectively, which is explicitly distinct from intracellular environment.

[0140] The term “Fig.” refers to its common meaning as an abbreviation for “Figure”, such that “Fig.1” represents Figure 1 which bears the same meaning as “FIG. 1”, so on and so forth throughput this disclosure.

[0141] The term “tumor associated antigen” (TAA) refers to a molecule with elevated expression in tumor cells as opposed to non-tumorous cells, such a molecule can be a protein or non-protein molecule, a peptide, a glycan, a glycolipid or a complex, including without limitation to HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSA, PSMA, AFP, STEAP1, STEAP2, STEAP3, STEAP4, KLK2, BCMA, GPRC5D, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1 (CD227), MUC16 (CA125), MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, FAP, Nectin-2, Nectin-4, B7-H3 (CD276), B7-H4, SLC34A2 (NaPi2b), AXL, Mer-TK, P-Cadherin, CDCP1, CDH3, CDH6, CDH17, CD25, CD33, CD38, CD46 (MCP), CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205 (Ly75), CD228 (SEMF), CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, PD-L1, ROR1, ROR2, ADAM17, FOLRl (FRα), Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), GUCY2C, CAIX (CA9), EphA2, gpA33, IGF1R, 29sf-6766980Docket No. 20222-20003.40 PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6 (LIV1A), TENB2, AGS-16, GPNMB, 17-A1 (CYP17A1), NKG2D, GD2, FcRH5, Fibronectin, CTHRC1, HLA-G, HLA-E, peptide-HLA-E, Tn glycan, and a mutated variant of NY-ESO, TP53, BRAF, N-RAS, H-RAS or K-RAS (e.g., K-RASG12D, K-RASG12V) or any of the mutant-derived peptides thereof, and a peptide-HLA complex overexpressed in a tumor in comparison to most normal tissues. Overexpression of some of the foregoing listed TAAs have been associated with one or more types of human cancers as reviewed previously (see Hafeez et al, Molecules, 25(20):4764 (2020)).

[0142] The term “CD3xTAA” or “CD3 x TAA” refers to its common meaning as an abbreviation for a multispecific (e.g., bispecific, trispecific, tetraspecific) antibody that comprises an anti-CD3 antigen-binding domain and an anti-TAA antigen-binding domain. Herein and thereafter and throughout this disclosure, “CD3xTAA” and “TAAxCD3” shall bear the same meaning and can be used interchangeably. As such, “CD3xTROP2” shall mean the same as “CD3 x TROP2”, “TROP2xCD3” and / or “TROP2 x CD3”, all referring to the same and one multispecific antibody that recognizes TROP2 and CD3.

[0143] The term “CD3 x TAA IgG” refers to a multispecific CD3xTAA molecule that is formatted in an IgG format comprising a Fc that serves as a half-life extending domain, wherein the IgG is derived from IgG1, IgG2, IgG3, or IgG4, wherein the molecule may comprise one or two identical copies of anti-TAA (i.e., the anti-TAA can be monovalent or bivalent), or one or two identical copies of anti-CD3 (i.e., the anti-CD3 can be monovalent or bivalent). In cases wherein the wording “2+1” is included in the molecular name, it refers to a multispecific CD3xTAA molecule that comprises a monovalent anti-CD3 domain and bivalent anti-TAA domains (e.g., two identical copies of a same anti-TAA domain).

[0144] The term “anti” or “α”refers to its conventional meaning when used as a prefix to indicate an antibody or an antigen-binding domain for a certain target. For instance, “anti-CD3” or “anti CD3” would bear the same conventional meaning as “α CD3” or “αCD3”, i.e., an antibody or antigen-binding fragment that recognizes CD3. Likewise, “anti-TAA”, “anti TAA”, “α TAA” or “αTAA” would bear the same conventional meaning and can be used interchangeably, so on and so forth.

[0145] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly indicates otherwise. 30sf-6766980Docket No. 20222-20003.40 II. Anti-CD3 Constructs

[0146] Anti-CD3 constructs described herein comprise an acidic pH-selective anti-CD3 antibody moiety (such as any of the acidic pH-selective anti-CD3 Fab or scFv described in this invention). In some embodiments, the anti-CD3 constructs described herein comprise an multispecific anti-CD3 construct comprising an anti-CD3 that is sensitive to pH changes. The current application provides the key features and coding sequences of such anti-CD3 constructs, their exemplary molecular configurations and the uses thereof for disease intervention.

[0147] In general, acidic pH-selective anti-CD3 constructs are meant non-conventional T cell mediated multispecific antibodies that preferentially engage T cells via CD3 to achieve T cell activation for target cell killing under an acidic microenvironment (e.g., an environment with a pH of at about 6.4 to about 6.9). In comparison, currently available CD3-based T cell- mediated multispecific antibodies typically do not show acidic pH-selectivity and in some cases rather exhibit weaker T cell-mediated target cell killing activity under acidic conditions at about pH 6.4 to about pH 6.9 as compared to conditions at about pH 7.3 to about pH 7.5.

[0148] For clarity, the term “anti-CD3 TCE” used herein in the current disclosure can be used interchangeably with “multispecific anti-CD3 construct” or “multispecific anti-CD3 antigen-binding molecules”. The term “multispecific” includes (without limitation to) the conventional “bispecific”, “trispecific”, and “tetraspecific”.

[0149] As used herein, the term “T cell engagers” (TCE) or “T cell mediated multispecific antibody” (“TMA”) refers to a biologic molecule that comprises at least a first and a second binding module, wherein the first binding module is capable of binding to a first antigen or target, and the second binding module is capable of binding to a second antigen or target, or to a second epitope of the same first antigen or target. In some embodiments, TCEs refer to a bispecific construct or molecule comprising two binding module. In some embodiments, TCEs refer to a multi-specific binding molecule such as a binding molecule comprising three binding modules or a tetra-specific binding molecule comprising four unique binding modules. As used herein, the term “binding domain” can be used interchangeably with “binding moiety” or “binding fragment” or “binding subunit” or “binding arm” or “binding region” or “binding module”. In some embodiments, TCEs comprise a single fusion polypeptide, a dimeric 31sf-6766980Docket No. 20222-20003.40 complex consisting of two polypeptides, a trimeric complex consisting of three polypeptides, or a multimeric complex consisting of four or more polypeptides. In some embodiments, the binding domain is a polypeptide or a portion or fragment of it thereof. In some embodiments, the binding domain comprises two or more portions from two or more polypeptides respectively. In the case that the TCE is a bispecific molecule, the term “TCE” is interchangeable with “TMA” (T cell mediated bispecific antibody). In some embodiments, conventional TCEs shows negligible or no significant pH preferences in terms of engaging T cells.

[0150] In one aspect, a TCE can engage a T cell from one portion of the TCE molecule, and engage a disease antigen molecule on a disease antigen-positive cell (e.g., cancer cell) from another portion of the same TCE, wherein the antigen (target molecule) on the disease cell is assumed to serve as a mechanical anchor and provide recognition specificity for the TCE. Upon forming a molecule bridge and an immune synapse between the said target cell and the said T cell, the T cell may become activated, and subsequently allow a molecular signaling cascade to occur, ultimately leading to intracellular transcriptional alterations, and phenotypic and functional changes of the T cell. Typical phenotypic and functional changes of the T cells include upregulation of activation markers (e.g., CD69, CD25, CD137), the immune- checkpoint molecules (e.g., PD-1, LAG3, TIM3), secretion of cytokines (e.g., IL-2, IFNγ, TNFα, IL-6, IL-10, IL-1, IL-8, IL-15, MCP-1, CXCL10) and in some cases the upregulation or mobilization of cytotoxic T cells’ intrinsic “killing machinery” (e.g., the apoptotic perforins and granzymes, and death receptor ligands such as Fas ligands). In some embodiments, the activated T cells can kill the engaged disease target cell by locally delivering a payload of perforins and granzymes through cell-surface holes on the target cells. Such cell-surface holes are generated by perforins that are also locally and directionally delivered via a microtubule network from the engaged T cell to the target cells. Upon the target cell death, the molecule bridge may become disengaged, and the T cells can then move on to engage and kill another target cell bearing the target antigens.

[0151] In some embodiments, provided is an anti-CD3 construct that comprises an anti-CD3 antibody domain based on any of the anti-CD3 antibody sequences described in this disclosure, optionally plus at least a second antigen-binding domain that binds an antigen on a disease cell such as a cancer cell. 32sf-6766980Docket No. 20222-20003.40

[0152] As exemplified in Fig. 1, the acidic pH-selective anti-CD3 constructs can be a bispecific molecule comprising a first antigen-binding domain that is an acidic pH-selective anti-CD3 binding domain, and a second antigen-binding domain that binds a disease antigen (e.g., tumor associated antigen). In some embodiments, the anti-CD3 constructs comprise one acidic pH-selective anti-CD3 binding domain and two anti-disease-antigen binding domains. In some embodiments, the anti-CD3 constructs comprise one acidic pH-selective anti-CD3 binding domain and at least two different anti-disease-antigen domains. In some embodiments, the anti-CD3 binding domain is formatted in a Fab or a scFv. In some embodiments, the anti- disease-antigen domain (e.g., anti-TAA) is formatted as a Fab, a scFv, a single-domain antibody, a nanobody, a VHH-only domain or an alternative antibody-mimicking scaffold. In some embodiments, the anti-CD3 constructs comprise two or more anti-disease-antigen domains that bind to the same epitope of a same target molecule. In some embodiments, the anti-CD3 constructs comprise two or more anti-disease-antigen domains that bind different epitopes respectively located on a same or two different disease antigen(s). In some embodiments, the acidic pH-selective anti-CD3 construct comprises a Fc or a Fc-like domain that is derived from an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgD or an IgE of human origin.

[0153] Illustrated in Fig. 2 are additional exemplary molecular configurations of acidic pH- selective anti-CD3 constructs. The anti-CD3 constructs can be a bispecific molecule comprising two acidic pH-selective anti-CD3 binding domains and two anti-TAA domains. The anti-CD3 constructs can be in a symmetrical (Fig. 2A) or an asymmetrical molecule configuration (Fig. 2B, 2C and 2D). The anti-TAA domain can be fused to the N-terminal region or the C-terminal region of an IgG light chain or heavy chain respectively (Fig.2A-2D). In some embodiments, the anti-CD3 binding domain is a Fab or a scFv. In some embodiments, the anti-disease-antigen domain (e.g., an anti-TAA domain) is a Fab, a scFv, a single-domain antibody, a nanobody, a VHH-only domain, a fragment of a cell surface receptor, or an alternative non-antibody scaffold. In some embodiments, the acidic pH-selective anti-CD3 construct comprises a Fc or a Fc-like domain that is derived from an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgD or an IgE of human origin.

[0154] In some embodiments, as exemplified in Fig.3, the anti-CD3 multispecific constructs comprise a single polypeptide comprising an acidic pH-dependent anti-CD3 antibody moiety 33sf-6766980Docket No. 20222-20003.40 and an anti-disease-antigen moiety such as an anti-TAA moiety. The anti-CD3 constructs can be a bispecific or trispecific molecule, comprising one acidic pH-selective anti-CD3 binding domain and one, two, or three domains that bind one disease target or two distinct disease targets. In some embodiments, the anti-CD3 constructs optionally comprises a half-life extending domain (half-life extender) that is derived from an anti-human serum albumin antibody (anti-HSA). Such a “half-life extending” domain can also be an antibody fragment that recognizes human transferrin, or an artificial polypeptide linker that is rich in glycine and serine residues.

[0155] In general, as illustrated in Fig. 4, an acidic pH-selective anti-CD3 constructs can be an a multispecific molecule that can bind CD3 in an acidic pH-biased manner to link T cells to a cancer cell for direct killing of the engaged cancer cell, e.g., in a tumor acidic microenvironment. The said anti-CD3 constructs can also in an acidic pH-biased manner induce cytokine secretion initiated from the engaged T cells, which can in turn activate or promote local bystander immune cells to mount a collectively enhanced or durable immune response against the tumor cells. Such mounted immune response may include additional cytokine release from the bystander immune cells proximal or local to the anti-CD3 construct- bound T cells. In some embodiments, the bystander cells can be T cells such as tumor infiltrating T cells and tissue resident T cells. In some embodiments, the bystander cells can be innate immune cells such as dendritic cells, NK cells, monocytes, neutrophils, basophils, eosinophils, and macrophages. In some embodiments, the bystander cells can be adaptive immune cells such as B cells.

[0156] On the other hand (shown in Fig. 4), in a healthy tissue environment that usually has a pH of about 7.3, 7.4 or 7.5, the acidic pH-selective anti-CD3 constructs will barely or only minimally engage and activate T cells and therefore will barely or only minimally trigger any significant killing of, and / or any significant cytokine release towards healthy cells that may express a target antigen at a physiological level.

[0157] In some embodiments, the target cells for the anti-CD3 constructs are tumor cells. In some embodiments, the target cells are cells with an abnormal proliferation property exemplified by a neoplasia or dysplasia pathological condition. In some embodiments, the target cells are general kinds of “disease associated cells”, which are not tumoral per se but nonetheless directly contribute to a disease condition. Exemplary non-tumoral disease cells 34sf-6766980Docket No. 20222-20003.40 include but are not limited to the following: (a) myeloid derived immune suppress cells (MDSCs) and regulatory T cells (Treg), each type of which is known to contribute to an immune-suppressive tumor microenvironment, (b) virus-infected immune or non-immune cells, (c) autoimmunity disorder associated disease cells, (d) inflammatory condition associated disease cells, and (e) tumor promoting cells such as M2 macrophages, M2-like macrophages, and / or stromal cells represented by cancer associated fibroblasts (CAFs). Generally, a disease cell associated target molecule can be a marker that is preferentially upregulated or overexpressed in the said disease associated cell. Exemplary disease cell associated targets are various tumor associated antigens for a variety of types of tumors, CD33 for myeloid derived suppressive cells, and virus (e.g., HPV, EBV, HSV, CMV, HIV, RSV) derived peptides that are presented on the surface of infected human cells via an MHC-I or MHC-II complex. As used herein, the term “tumor associated antigen” or “TAA” shall generally include the so-called “tumor neoantigen” and “tumor specific antigen”.

[0158] In some embodiments, the anti-TAA antigen-binding fragment can be selected from the following list: (1) antibodies and antibody-like molecules, and further derivative or fusion forms of these molecules thereof; (2) cytokine or cytokine-like molecules, including cytokines, growth factors, chemokines, extra-cellular domains of cell membrane associated proteins (e.g., cell surface receptors), and the further derivative or fusion forms of these said molecules thereof; (3) alternative non-antibody scaffolds; (4) any combination or pairing from (1), (2) and (3) in the foregoing sentence with exemplary examples of antibody-cytokine fusions or immunocytokines and their various forms of derivatives and modifications.

[0159] In some embodiments, as exemplified in Fig. 3, the anti-CD3 construct comprises a single polypeptide chain comprising one pH-sensitive anti-CD3 fragment and one anti-disease antigen fragment, wherein the two fragments are fused by a linker. Optionally, the single polypeptide chain of the anti-CD3 construct further comprises a domain that serves to extend the half-life (or pharmacokinetics) of the construct. Such “half-life extending” domains are exemplified by a single domain antibody recognizing human serum albumin (HSA) or transferrin.

[0160] It is generally understood for a person skilled in the art that a target antigen-binding moiety can be an antibody domain selected from a group consisting of a scFv (i.e., single-chain variable fragment), a Fab, a Fab’, a F(ab’)2, a single domain antibody (sdAB), a CrossMab, a 35sf-6766980Docket No. 20222-20003.40 VHH-only domain, a nanobody, an IgG heavy chain, an IgG light chain, and an engineered or a combination of these antibody-derivative molecules thereof. In another aspect, it is also understood that a target antigen-binding moiety can be based on an alternative non-antibody scaffold that is selected from a group consisting of a cell receptor fragment, an Affibody, an Affilin, an Alphabody, a Knottin, a DARPin, an Anticalin, a Kunitz domain peptide, a FN3 scaffold, a Fynomer, a Cys-knots, a Monobody, an Affimer, a peptide-MHC polypeptide, a lectin or lectin-derived domain and a non-natural or semi-natural synthetic peptide or polypeptide.

[0161] In some embodiments, the two or more antigen-binding moieties comprised by acidic pH-selective anti-CD3 constructs are linked or fused to form an integral molecule through a linker (such as any one of the linkers described herein). In some embodiments, the linker is a peptide linker or a chemical linker. Exemplary peptide linkers are: (1) a flexible peptide linker with a length of about 1 to about 49 amino acids, represented by a glycine / serine rich linker such as (GS)n(SEQ ID NO: 181), (GGS)n(SEQ ID NO: 182), (GGGS)n(SEQ ID NO: 185), (GGGGGS)n (SEQ ID NO: 179), (GGSG)n (SEQ ID NO: 178), (GGGGS)n (SEQ ID NO: 186), (GGSGG)n (SEQ ID NO: 177), wherein n is an integer between 1 and 20; (2) a rigid peptide linker with a length of about 1 to about 50 amino acids, represented by a E / A / P rich linker such as A(EAAAK)n A (SEQ ID NO: 176) and T(A4T)n AAA (SEQ ID NO: 175), wherein n is an integer between 1 and 9; (3) a peptide linker derived from a natural protein, such protein represented by FNRGEC (SEQ ID NO: 174) and EPSGP (SEQ ID NO: 173) (derived from an IgG upper hinge region), LGGC (SEQ ID NO: 172) and VEPKEC (SEQ ID NO: 171) (derived from the C-terminus of the kappa light chain of an IgG molecule), and PSGQAGAAASESLFVSNHAY (SEQ ID NO: 170) (derived from human muscle aldolase); (4) a combination of any of the foregoing described (1) and (2) peptide linkers of various compositions and biophysical properties. In some embodiments, the linkers are chemical linkers, such as polyethylene glycol (PEG), polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), and poly(ɛ-caprolactone) (PCL). In some embodiments, the preferred linker is a peptide linker with a length of about 2 to about 50 amino acids. It is understood that the composition, length, stiffness, flexibility, orientation, and charge of the linker is often crucial for the proper assembling and functional activity of bispecific or multispecific molecules including . 36sf-6766980Docket No. 20222-20003.40

[0162] In some embodiments, the pH-selective anti-CD3 construct is a multispecific molecule that comprises a dimerization domain such as an engineered heterodimerizing Fc domain derived from an IgG, or a trimerization domain such as a Dock-and-Lock (DNL) domain (e.g., a leucine zipper derived from cAMP-dependent protein kinase (PKA) and A kinase anchor proteins (AKAPs). In some embodiments, the heterodimerizing Fc domain is achieved by introducing asymmetric mutations in each of CH2 and / or CH3 domain respectively, which favors the assembly of a heterodimeric Fc rather than a conventional homodimer Fc. These asymmetric mutations either form hydrophobic / steric complementarity (e.g., mutations in the so-called Knobs-into-Holes Fc format, or “KIH” for brevity), electrostatic complementarity (e.g., DD vs KK variations), complementarity from alternating sequences from IgG and IgA (e.g., mutations in the so-called SEED format) or stable complementarity through charge and hydrogen bonding (e.g. mutations in the so-called XmAb format). Other types of heterodimerizations are achieved through purification by introducing mutations that abolish protein binding (e.g. so-called Veloci-Bi or BEAM technologies).

[0163] To achieve correct pairing of light chains (LC) from two different antibodies, heterodimeric heavy chain bispecific antibodies have been developed in combination with a common LC approach or with two distinct LCs, using the CrossMab technology with only the VH and VL domains crossover, using FIT-IgG technology with Fab domains crossover, using engineered interface between CH / VL, using the so-called WuXiBody technology by replacing CK / CH1 with TCR alpha and beta constant regions, using EFab domain technology by replacing one of CK and CH1 with the CH2 of IgE.

[0164] Heterodimerization between antibody moieties can be achieved using other variants of engineered human IgG1 Fc domain that favors heterodimerization, for instance, Alphamab’s bispecific Fc platform “CRIB” (Charge Repulsion Induced Bispecific), Merus’ bispecific Fc platform “Biclonics” that bears L351D / L368E changes in Fc chain 1 and L351K / T366K changes in Fc chain 2, Zymework’s bispecific Fc domain that harbors T350V / T366L / K392L / T394W mutations in Fc chain 1 and T350V / L351Y / F405A / Y407V in Fc chain 2, Glenmark’s “BEAT” bispecific Fc platform, and other variants of Knobs-into- Holes (“KIH” or “KiH”).

[0165] In certain embodiments, the anti-CD3 construct or polypeptide complex is formatted as a bispecific molecule selected from a variety of established bispecific antibody platforms, 37sf-6766980Docket No. 20222-20003.40 including but not limited to: Triomabs; the anticalin-based multispecific platform (Pieris); Diabodies; single-chain diabodies; tandem single-chain Fv fragments; TandAbs and trispecific antibodies (Affimed); DARTs (Dual Affinity ReTargeting; Macrogenics); bispecific XmAbs (Xencor); bispecific T cell engagers (BiTEs; Amgen); triplebodies; the DuoBody platform (Genmab); Knobs-into-Holes (KiH); humanized bispecific IgG antibodies (e.g., REGN1979, Regeneron); Mab2 bispecifics (F-star); dual-variable domain immunoglobulins (DVD-Ig; AbbVie); kappa-lambda bodies; tetravalent bispecific tandem Ig (TBTI); and CrossMab technology (Roche).

[0166] In other embodiments, the anti-CD3 construct or polypeptide complex is based on a bispecific IgG-like antibody (BsIgG) architecture, selected from formats including: CrossMab; dual-affinity formats (DAF, both two-in-one and four-in-one); DutaMab; WuXiBody; DT-IgG; Knobs-in-Holes with common light chain; Knobs-in-Holes with separate assembly strategies; charge pair-based designs; Fab-arm exchange; SEEDbodies; Triomabs; LUZ-Y; Fcabs; kappa- lambda bodies; and Orthogonal Fab designs. For further technical detail on these formats, see Spiess C., Zhai Q., and Carter P.J. (2015), Molecular Immunology, 67:95–106, which is hereby incorporated by reference in its entirety.

[0167] In some embodiments, the bispecific polypeptide complex comprises IgG-appended antibodies featuring additional antigen-binding domains. Representative formats include: DVD-IgG; IgG(H)-scFv; scFv-IgG(H); IgG(L)-scFv; scFv-IgG(L); IgG(L,H)-Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KiH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; Zybody; and DVI-IgG (four-in-one format) (see id.).

[0168] In further embodiments, the bispecific construct is based on antibody fragment formats, including: Nanobody and Nanobody-HAS fusions; BiTEs; Diabodies; DARTs; TandAbs; single-chain diabodies (scDiabodies); sc-Diabody-CH3; Diabody-CH3; triple bodies; miniantibodies; minibodies; TriBi minibodies; scFv-CH3 with KiH; Fab-scFv fusions; scFv-CH–CL-scFv; F(ab′)₂; F(ab′)₂-scFv₂; scFv-KiH; Fab-scFv-Fc fusions; tetravalent heavy- chain antibodies (HCAb); scDiabody-Fc; Diabody-Fc; tandem scFv-Fc fusions; and intrabodies. 38sf-6766980Docket No. 20222-20003.40 Anti-CD3 antibody moieties

[0169] The present disclosure provides various innovative anti-CD3 constructs that comprise an antibody moiety that binds both human and monkey CD3, comprising a heavy variable region (VH) and a light chain variable region (VL).

[0170] The disclosed anti-CD3 antibodies can incorporate amino acid substitutions, insertions, and / or deletions within the framework and / or CDR regions of the heavy chain variable domains compared to the respective germline sequences. The disclosure further encompasses antibodies and antigen-binding fragments derived from any of the disclosed amino acid sequences, wherein one or more amino acids within one or more framework, CDR regions, VHand / or VLundergo mutation to the corresponding residue(s) of the germline sequence of origin or another human germline sequence, or undergo a conservative amino acid substitution of the corresponding germline residue(s), resulting in enhanced or reduced binding to a CD3 antigen, or leading to improved pharmacological property, functional property, and / or reduced immunogenicity of the anti-CD3 antibody or antibodies.

[0171] In some embodiments, as exemplified in Tables 3 and 4, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 63, the LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 65, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 130, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 150, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0172] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, the HC-CDR2 comprising the amino 39sf-6766980Docket No. 20222-20003.40 acid sequence of SEQ ID NO: 5, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 66, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 67, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 68, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 131, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 151, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0173] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 9, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 69, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 70, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 132, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 152, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0174] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 10, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the 40sf-6766980Docket No. 20222-20003.40 amino acid sequence of SEQ ID NO: 72, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 73, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 133, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 153, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0175] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 75, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 76, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 77, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 134, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 154, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0176] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 16, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 78, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 79, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 80, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- 41sf-6766980Docket No. 20222-20003.40 CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 135, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 155, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0177] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 81, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 82, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 83, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 136, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 156, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0178] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 22, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 24, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 84, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 85, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 86, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 137, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL42sf-6766980Docket No. 20222-20003.40 comprises an amino acid sequence of SEQ ID NO: 157, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0179] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 25, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 87, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 88, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 89, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 138, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 158, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0180] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 28, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 30, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 90, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 371, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 471, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity. 43sf-6766980Docket No. 20222-20003.40

[0181] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 93, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 94, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 95, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 372, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 472, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0182] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 34, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 96, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 97, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 98, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 373, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 473, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0183] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 37, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and the HC-CDR3 comprising the amino acid sequence of 44sf-6766980Docket No. 20222-20003.40 SEQ ID NO: 39, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 99, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 100, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 101, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 374, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 474, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0184] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 102, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 103, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 375, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 475, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0185] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 43, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 105, the LC-CDR2 comprising the amino acid sequence 45sf-6766980Docket No. 20222-20003.40 of SEQ ID NO: 106, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 107, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 376, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 476, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0186] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 46, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 108, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 109, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 110, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 377, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 477, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0187] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 49, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 51, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 111, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 113, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 378, 46sf-6766980Docket No. 20222-20003.40 or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 478, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0188] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 52, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 114, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 115, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 116, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 379, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 479, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0189] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 57, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 117, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 118, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 119, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 380, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 480, or a variant comprising an amino acid 47sf-6766980Docket No. 20222-20003.40 sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0190] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 58, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 301, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 401, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 402, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 403, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 381, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 481, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0191] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 302, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 303, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 304, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 404, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 405, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 406, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 382, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 482, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity. 48sf-6766980Docket No. 20222-20003.40

[0192] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 305, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 306, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 307, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 407, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 408, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 409, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 383, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 483, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0193] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 308, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 309, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 310, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 410, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 411, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 412, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 384, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 484, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0194] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 311, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 312, and the HC-CDR3 comprising the amino acid sequence of 49sf-6766980Docket No. 20222-20003.40 SEQ ID NO: 313, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 413, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 414, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 415, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 385, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 485, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0195] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 314, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 315, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 316, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 416, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 417, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 418, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 386, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 486, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0196] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 317, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 318, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 319, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 419, the LC-CDR2 comprising the amino acid sequence 50sf-6766980Docket No. 20222-20003.40 of SEQ ID NO: 420, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 421, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 387, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 487, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0197] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 320, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 321, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 322, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 422, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 423, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 424, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 388, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 488, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0198] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 323, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 324, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 325, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 425, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 426, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 389, 51sf-6766980Docket No. 20222-20003.40 or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 489, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0199] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 326, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 327, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 328, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 428, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 429, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 430, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 390, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 490, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0200] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 329, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 330, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 331, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 431, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 432, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 433, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 391, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 491, or a variant comprising an amino acid 52sf-6766980Docket No. 20222-20003.40 sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0201] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 332, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 333, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 334, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 434, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 435, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 436, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VHcomprises an amino acid sequence of SEQ ID NO: 392, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 492, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0202] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 335, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 336, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 337, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 437, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 438, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 439, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 393, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 493, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity. 53sf-6766980Docket No. 20222-20003.40

[0203] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 338, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 339, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 340, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 440, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 441, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 442, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 394, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VLcomprises an amino acid sequence of SEQ ID NO: 494, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0204] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 341, the HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 342, and the HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 343, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 443, the LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 444, and the LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 445, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC- CDRs. In some embodiments, the VH comprises an amino acid sequence of SEQ ID NO: 395, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity; and the VL comprises an amino acid sequence of SEQ ID NO: 495, or a variant comprising an amino acid sequence having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.

[0205] In some embodiments, the CDRs of anti-CD3 antibodies comprises three HC-CDRs and three LC-CDRs comprising the sequences of SEQ ID NOs: 1 through 3 and SEQ ID NOs: 63 through 65 respectively, or comprising the sequences of SEQ ID NOs: 4 through 6 and SEQ 54sf-6766980Docket No. 20222-20003.40 ID NOs: 66 through 68 respectively, or comprising the sequences of SEQ ID NOs: 7 through 9 and SEQ ID NOs: 69 through 71 respectively, or comprising the sequences of SEQ ID NOs: 10 through 12 and SEQ ID NOs: 72 through 74 respectively, or comprising the sequences of SEQ ID NOs: 13 through 15 and SEQ ID NOs: 75 through 77 respectively, or comprising the sequences of SEQ ID NOs: 16 through 18 and SEQ ID NOs: 78 through 80 respectively, or comprising the sequences of SEQ ID NOs: 19 through 21 and SEQ ID NOs: 81 through 83 respectively, or comprising the sequences of SEQ ID NOs: 22 through 24 and SEQ ID NOs: 84 through 86 respectively, or comprising the sequences of SEQ ID NOs: 25 through 27 and SEQ ID NOs: 87 through 89 respectively, or comprising the sequences of SEQ ID NOs: 28 through 30 and SEQ ID NOs: 90 through 92 respectively, or comprising the sequences of SEQ ID NOs: 31 through 33 and SEQ ID NOs: 93 through 95 respectively, or comprising the sequences of SEQ ID NOs: 34 through 36 and SEQ ID NOs: 96 through 98 respectively, or comprising the sequences of SEQ ID NOs: 37 through 39 and SEQ ID NOs: 99 through 101 respectively, or comprising the sequences of SEQ ID NOs: 40 through 42 and SEQ ID NOs: 102 through 104 respectively, or comprising the sequences of SEQ ID NOs: 43 through 45 and SEQ ID NOs: 105 through 107 respectively, or comprising the sequences of SEQ ID NOs: 46 through 48 and SEQ ID NOs: 108 through 110 respectively, or comprising the sequences of SEQ ID NOs: 49 through 51 and SEQ ID NOs: 111 through 113 respectively, or comprising the sequences of SEQ ID NOs: 52 through 54 and SEQ ID NOs: 114 through 116 respectively, or comprising the sequences of SEQ ID NOs: 55 through 57 and SEQ ID NOs: 117 through 119 respectively, or comprising the sequences of SEQ ID NOs: 58, 59, 301, and SEQ ID NOs: 401 through 403 respectively, or comprising the sequences of SEQ ID NOs: 302 through 304 and SEQ ID NOs: 404 through 406 respectively, or comprising the sequences of SEQ ID NOs: 305 through 307 and SEQ ID NOs: 407 through 409 respectively, or comprising the sequences of SEQ ID NOs: 308 through 310 and SEQ ID NOs: 410 through 412 respectively, or comprising the sequences of SEQ ID NOs: 311 through 313 and SEQ ID NOs: 413 through 415 respectively, or comprising the sequences of SEQ ID NOs: 314 through 316 and SEQ ID NOs: 416 through 418 respectively, or comprising the sequences of SEQ ID NOs: 317 through 319 and SEQ ID NOs: 419 through 421 respectively, or comprising the sequences of SEQ ID NOs: 320 through 322 and SEQ ID NOs: 422 through 424 respectively, or comprising the sequences of SEQ ID NOs: 323 through 325 and SEQ ID NOs: 425 through 427 respectively, 55sf-6766980Docket No. 20222-20003.40 or comprising the sequences of SEQ ID NOs: 326 through 328 and SEQ ID NOs: 428 through 430 respectively, or comprising the sequences of SEQ ID NOs: 329 through 331 and SEQ ID NOs: 431 through 433 respectively, or comprising the sequences of SEQ ID NOs: 332 through 334 and SEQ ID NOs: 434 through 436 respectively, or comprising the sequences of SEQ ID NOs: 335 through 337 and SEQ ID NOs: 437 through 439 respectively, or comprising the sequences of SEQ ID NOs: 338 through 340 and SEQ ID NOs: 440 through 442 respectively, or comprising the sequences of SEQ ID NOs: 341 through 343 and SEQ ID NOs: 443 through 445 respectively. In some embodiments, the CDRs of any of the anti-CD3 antibodies in this section comprises a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC-CDRs or comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDRs.

[0206] In some embodiments, the VHof anti-CD3 antibodies comprises a HC-CDR1 comprising the sequence selected from the list of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, and 341, a HC-CDR2 comprising a sequence selected from the list of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, and 342; and a HC-CDR3 comprising a sequence selected from the list of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, and 343. In some embodiments, the three HC-CDRs of any of the anti-CD3 antibodies in this section comprises a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC- CDRs.

[0207] In some embodiments, the VH of anti-CD3 antibodies comprises a HC-CDR1 comprising a sequence selected from the list of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 302, 305, 308, 311, 314, 317, 320, 323, 326, 329, 332, 335, 338, and 341, a HC-CDR2 comprising a sequence selected from the list of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 303, 306, 309, 312, 315, 318, 321, 324, 327, 330, 333, 336, 339, and 342; and a HC-CDR3 comprising a sequence selected from the list of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 301, 304, 307, 310, 313, 316, 319, 322, 325, 328, 331, 334, 337, 340, and 343. In some embodiments, the three HC-CDRs of any of the anti-CD3 antibodies in this section 56sf-6766980Docket No. 20222-20003.40 comprises a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the HC- CDRs.

[0208] In some embodiments, the VL of anti-CD3 antibodies comprises a LC-CDR1 comprising a sequence selected from the list of SEQ ID NOs: 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 401, 404, 407, 410, 413, 416, 419, 422, 425, 428, 431, 434, 437, 440 and 443, a LC-CDR2 comprising a sequence selected from the list of SEQ ID NOs: 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 402, 405, 408, 411, 414, 417, 420, 423, 426, 429, 432, 435, 438, 441, and 444; and a LC-CDR3 comprising a sequence selected from the list of SEQ ID NOs: 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 403, 406, 409, 412, 415, 418, 421, 424, 427, 430, 433, 436, 439, 442, and 445. In some embodiments, the three LC-CDRs of any of the anti- CD3 antibodies in this section comprises a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions in the LC-CDRs.

[0209] In some embodiments, the anti-CD3 antibody comprises a VHcomprising the amino acid sequence selected from the list of SEQ ID NOs: 130, 131, 132, 133, 134, 135, 136, 137, 138, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, and 395, or a variant having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the foregoing listed VH sequences in this paragraph, and further comprises a VL comprising the amino acid sequence selected from SEQ ID NOs: 150, 151, 152, 153, 154, 155, 156, 157, 158, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, or a variant having at least about 90% (such as about 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of the foregoing listed VL sequences in this paragraph.

[0210] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising the consensus amino acid sequence of SEQ ID NO: 60, the HC-CDR2 comprising the consensus amino acid sequence of SEQ ID NO: 61, and the HC-CDR3 comprising the consensus amino acid sequence of SEQ ID NO: 62, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid alterations the HC-CDRs, and the VL of anti-CD3 antibodies comprises the LC-CDR1 comprising the consensus amino acid sequence of SEQ ID NO: 120, the LC- CDR2 comprising the consensus amino acid sequence of SEQ ID NO: 121, and the LC-CDR3 57sf-6766980Docket No. 20222-20003.40 comprising the consensus amino acid sequence of SEQ ID NO: 122, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid alterations in the LC-CDRs.

[0211] In some embodiments, the VH of anti-CD3 antibodies comprises a HC-CDR1 sequence selected from SEQ ID NOs: 1, 22, 43, 49, and 326, a HC-CDR2 sequence selected from SEQ ID NOs: 2, 5, 47, 324, 327, 330, and 333, and a HC-CDR3 sequence selected from SEQ ID NOs: 3, 6, 9, 15, 337, 340 and 343, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid alterations in any of the three HC-CDRs, and the VLof anti-CD3 antibodies comprises a LC-CDR1 sequence selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99, and 413, a LC-CDR2 sequence selected from SEQ ID NOs: 64, 417 and 420, and a LC-CDR3 sequence corresponding to SEQ ID NO: 65, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid alterations in any of the three LC-CDRs.

[0212] In some embodiments, the VH of anti-CD3 antibodies comprises a HC-CDR1 sequence selected from SEQ ID NOs: 1, 22, 43, and 49, a HC-CDR2 sequence selected from SEQ ID NOs: 5, 47, 324, and 330, and a HC-CDR3 sequence selected from SEQ ID NOs: 6, 9, 15, 337, 340 and 343, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid alterations in any of the three HC-CDRs, and the VL of anti-CD3 antibodies comprises a LC- CDR1 sequence selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99, and 413, a LC-CDR2 sequence selected from SEQ ID NOs: 64, 417 and 420, and a LC-CDR3 sequence corresponding to SEQ ID NO: 65, or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid alterations in any of the three LC-CDRs.

[0213] In some embodiments, the VHof anti-CD3 antibodies comprises the HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 60, the HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and the HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 207, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 120, the LC-CDR2 comprising an amino acid sequence of SEQ ID NO: 121, and the LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 122. In some embodiments, the LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 66, 69, 75, 81, 87 or 99.

[0214] In some embodiments, the VH of anti-CD3 antibodies comprises the HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 4, the HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 5, and the HC-CDR3 comprising an amino acid sequence of 58sf-6766980Docket No. 20222-20003.40 SEQ ID NO: 207, and the VLof anti-CD3 antibodies comprises the LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 120, the LC-CDR2 comprising an amino acid sequence of SEQ ID NO: 121, and the LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 122. In some embodiments, the LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 66, 69, 75, 81, 87 or 99.

[0215] As is known to those skilled in the art, the framework sequence of the variable regions of an antibody are often inter-changeable with one or more conserved germline framework sequences with minimal or no impact on the antibody’s biological activity. In some embodiments, the VH framework of the anti-CD3 variant (corresponding to SEQ ID NOs: 130, 131, 132, 133, 134, 135, 136, 137, 138, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394 or 395) is derived from the human VH germline alleles hIGHV1-3*01, hIGHV1-2*02, hIGHV1-2*03, or hIGHV1-2*04. In some embodiments, the VL framework of the anti-CD3 variant (corresponding to SEQ ID NOs: 150, 151, 152, 153, 154, 155, 156, 157, 158, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, or 488) is derived from the human VL germline alleles hIGKV4-1*01, hIGKV2D-28*01, hIGKV2-28*01, hIGKV2D-29*02.

[0216] In some embodiments, the anti-CD3 antibody comprises a VHcomprising a HC- CDR1 sequence selected from SEQ ID NOs: 1, 22, 43, 49, and 326, a HC-CDR2 sequence selected from SEQ ID NOs: 2, 5, 47, 324, 327, 330 and 333, a HC-CDR3 sequence selected from SEQ ID NOs: 3, 6, 9, 15, 337, 340 and 343, and a framework derived from hIGHV1- 3*01, hIGHV1-2*02, hIGHV1-2*03, or hIGHV1-2*04, and further comprises a VLcomprising a LC-CDR1 sequence selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99 and 413, a LC-CDR2 sequence selected from SEQ ID NOs: 64, 417 and 420, a LC-CDR3 sequence corresponding to SEQ ID NO: 65, and a framework derived from hIGKV4-1*01, hIGKV2D- 28*01, hIGKV2-28*01, or hIGKV2D-29*02.

[0217] In some embodiments, the anti-CD3 antibody comprises a VH comprising a HC- CDR1 sequence selected from SEQ ID NOs: 1, 22, 43, 49, and 326, a HC-CDR2 sequence selected from SEQ ID NOs: 5, 47, 324 and 330, a HC-CDR3 sequence selected from SEQ ID NOs: 6, 9, 15, 337, 340 and 343, and a framework derived from hIGHV1-3*01, hIGHV1-2*02, or hIGHV1-2*03, and further comprises a VLcomprising a LC-CDR1 sequence selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99 and 413, a LC-CDR2 sequence selected from SEQ ID 59sf-6766980Docket No. 20222-20003.40 NOs: 64, 417 and 420, a LC-CDR3 sequence corresponding to SEQ ID NO: 65, and a framework derived from hIGKV4-1*01 or hIGKV2D-28*01.

[0218] In some embodiments, the amino acid substitutions in any one of the disclosed anti- CD3 antibody sequences are limited to “exemplary substitutions” shown in Table 2 of this disclosure. In some embodiments, the amino acid substitutions are based on “preferred substitutions” shown in Table 2 of this disclosure. The amino acid alterations can be substitutions, deletions, insertions, or their combinations or hybrids thereof.

[0219] The HC-CDRs amino acid sequences of disclosed exemplary pH-selective anti-CD3 antibody moieties are shown Table 3. Table 3. VHCDRs of various pH-selective anti-CD3 antibodies and consensus sequences.60sf-6766980Docket No. 20222-20003.4061sf-6766980Docket No. 20222-20003.40

[0220] The LC-CDRs amino acid sequences of disclosed exemplary pH-selective anti-CD3 antibody moieties are shown Table 4. Table 4. VLCDRs of various pH-selective anti-CD3 antibodies and consensus sequences.62sf-6766980Docket No. 20222-20003.4063sf-6766980Docket No. 20222-20003.40 a) Antibody affinity

[0221] Binding specificity of the antibody moieties can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA, RIA, ECL, IRMA, EIA, Flow cytometry (FC), Bio-Layer Interferometry (e.g., Octet®BLI and Gator®BLI), Surface Plasma Resonance (e.g., Biacore®SPR) and peptide scans.

[0222] As shown in ELISA assays (Fig. 5), representative variants of acidic pH-biased anti- CD3 antibodies such as h10.v52 and h10.v63 (the same as disclosed in the Sequence Table) can bind to CD3 in a pH dependent manner, exhibiting much stronger binding under acidic conditions with a pH of about 6.0 to about 6.7, and relatively weaker binding under physiologically-relevant condition with a pH of about 7.3 to about 7.5. In contrast, the non-pH- sensitive clone h10.v65 shows minimal change of its binding activity towards CD3 under acidic or near physiological pH conditions.

[0223] In some embodiments, the acidic pH-selective anti-CD3 antibody, such as any one of the anti-CD3 variants exemplified in Sequence Table by h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261 and h10.v263, has a medium-to-high monovalent affinity for CD3 (e.g., human CD3E) with a KDranging from about 0.1 nM to about 100 nM, or from about 1 nM to about 50 nM under conditions with a pH of about 6.0 to about 6.6. In contrast, under conditions with a pH of about 7.4, the same anti-CD3 antibody has a much-reduced monovalent affinity for CD3 with a KDranging from at least about 10 nM, about 15 nM, about 20 nM, about 25 nM, or about 30 nM to a near undetectable high level, or from about 70 nM to about 4 µM. In some embodiments, the said anti-CD3 antibody has a monovalent affinity KD for CD3 under assay conditions of about pH 7.4, that is at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 8-fold, or at least about 10-fold as that detected under assay conditions of about pH 6.0 to about 6.6. In some embodiments, the CD3 is human CD3E or a complex comprising human CD3E; in other words, the anti-CD3 antibody has an affinity-based functional pH selectivity index (“affinity FPSI”) of at least 3. In some 64sf-6766980Docket No. 20222-20003.40 embodiments, the affinity FPSI (e.g., KDratio of pH 7.4 vs. pH 6.6) is at least 3, 4, 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, or 37. In some embodiments, the affinity FPSI (e.g., KD ratio of pH 7.4 vs. pH 6.6) is in the range of about 3 to about 40. In some embodiments, the affinity FPSI (e.g., KDratio of pH 7.4 vs. pH 6.6) is in the range of about 5 to about 40. In some embodiments, the affinity FPSI (e.g., KD ratio of pH 7.4 vs. pH 6.6) is in the range of about 10 to about 40. In some embodiments, the affinity FPSI (e.g., KDratio of pH 7.4 vs. pH 6.6) is in the range of about 15 to about 40. In some embodiments, the affinity FPSI (e.g., KD ratio of pH 7.4 vs. pH 6.6) is in the range of about 20 to about 40. In some embodiments, the affinity FPSI (e.g., KD ratio of pH 7.4 vs. pH 6.6) is in the range of about 25 to about 40. In some embodiments, the affinity FPSI (e.g., KDratio of pH 7.4 vs. pH 6.6) is in the range of about 30 to about 40.

[0224] In some embodiments, the Kon of the kinetic binding between the antibody moiety and CD3 is about 103M-1s-1to about 108M-1s-1, about 103M-1s-1to about 104M-1s-1, about 104M-1s-1to about 105M-1s-1, about 105M-1s-1to about 106M-1s-1, about 106M-1s-1to about 107M-1s-1, or about 107M-1s-1to about 108M-1s-1. In some embodiments, the Kon of the binding between the antibody moiety and CD3 is about 103M-1s-1to about 105M-1s-1, about 104M-1s-1to about 106M-1s-1, about 105M-1s-1to about 107M-1s-1, about 106M-1s-1to about 108M-1s-1, about 104M-1s-1to about 107M-1s-1, or about 105M-1s-1to about 108M-1s-1. In some embodiments, the Kon of the binding between the antibody moiety and CD3 is no more than about any one of 103M-1s-1, 104M-1s-1, 105M-1s-1, 106M-1s-1, 107M-1s-1or 108M-1s-1. In some embodiments, the CD3 is human or monkey CD3E or a complex comprising human or monkey CD3E.

[0225] In some embodiments, the Koff of the binding between the antibody moiety and CD3 is about 1 s-1to about 10-6s-1, about 1 s-1to about 10-2s-1, about 10-2s-1to about 10-3s-1, about 10-3s-1to about 10-4s-1, about 10-4s-1to about 10-5s-1, about 10-5s-1to about 10-6s-1, about 1 s-1to about 10-5s-1, about 10-2s-1to about 10-6s-1, about 10-3s-1to about 10-6s-1, about 10-4s-1to about 10-6s-1, about 10-2s-1to about 10-5s-1, or about 10-3s-1to about 10-5s-1. In some embodiments, the CD3 is human or monkey CD3E or a complex comprising human or monkey CD3E. 65sf-6766980Docket No. 20222-20003.40

[0226] The monovalent affinity KDvalues of anti-CD3 antibody variants for a recombinant human CD3E extracellular domain are summarized in Table 5. Binding affinity was assayed by a biolayer interferometry (BLI) method. Table 5. Affinity KD (nM) of anti-CD3 variants for human CD3E.66sf-6766980Docket No. 20222-20003.40

[0227] The monovalent affinity KDvalues of various anti-CD3 antibodies for a recombinant cynomolgus monkey CD3E extracellular domain are summarized in Table 6 (based on a BLI assay method). Table 6. Affinity KD (nM) of anti-CD3 variants for cynomolgus monkey CD3E.67sf-6766980Docket No. 20222-20003.40

[0228] In general, in order to enable monkey as a human-relevant toxicity study model, it may be desired that the fold difference with respect to the binding affinity for human CD3 and monkey CD3 is no more than 5 folds, no more than 4 folds, or preferentially no more than 3 folds. In some embodiments, any one of the disclosed acidic pH-selective anti-CD3 clones (h10.v52, h10.v63, h10.v80, h10.v84, h10.v154, h10.v168, h10.v170, h10.v176 or h10.v190) showed comparable kinetic affinity towards human and monkey CD3, with a fold difference of no more than 3 under a pH of about 6.6, wherein the binding is stronger than at about pH 7.4 and therefore can be detected relatively more accurately.

[0229] In some embodiments, as shown in flow cytometry-based cell binding assays (Fig. 6), pH selective anti-CD3 antibody variants (e.g., h10.v52 and h10.v63 in a Fab format) bind human T cells (human primary T cells) in a pH dependent manner, respectively, wherein the cell-surface binding (shown as mean fluorescent intensity) is the strongest at the lower end of the tested pH range (about pH 5.8-6.0), but is reduced upon pH increase and is the weakest at the higher end of the tested pH range (about pH 7.4) (Fig. 6A), showing about 22-fold and 15- fold decrease for h10.v52 and h10.v63 respectively (Fig. 6B). In comparison, the non-pH selective anti-CD3 clone h10.wt and h10.v65 shows a small change in its cell-surface binding to the same T cells under the serial pH titration (< 2.7-fold difference). As described herein, flow cytometry is a commonly used cell analysis technology that can be performed by an average skilled person using any one of the many commercially available flow cytometry instruments. a) Chimeric or humanized antibodies

[0230] In some embodiments, the antibody moiety is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from mouse) and a human constant region. In some embodiments, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0231] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the 68sf-6766980Docket No. 20222-20003.40 specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0232] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0233] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); Framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0234] It is understood that the humanization of mouse derived antibodies is a common and routinely used art. It is therefore understood that a humanized format of any and all of the anti- CD3 antibodies disclosed in Sequence Table can be used in a preclinical or clinical setting. In cases where a humanized format of any of the referenced anti-CD3 antibodies or their antigen- binding fragments thereof is used in such a preclinical or clinical setting, the then humanized 69sf-6766980Docket No. 20222-20003.40 format is expected to bear the same or similar biological activities and properties as the original non-humanized format. b) Substitution, insertion, deletion and variants

[0235] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs (or CDRs) and framework regions (FRs). Conservative substitutions are shown in Table 2 under the heading of “Preferred substitutions.” More substantial changes are provided in Table 2 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC properties. Table 2. Amino Acid Substitutions.70sf-6766980Docket No. 20222-20003.40

[0236] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0237] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0238] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, increased production yield, increased stability, reduced immunogenicity, increased solubility) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

[0239] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted. 71sf-6766980Docket No. 20222-20003.40

[0240] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or CDRs. In some embodiments of the variant VHH sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.

[0241] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0242] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. c) Glycosylation variants

[0243] In some embodiments, the antibody moiety is altered to increase or decrease the extent to which the construct is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. 72sf-6766980Docket No. 20222-20003.40

[0244] Where the antibody moiety comprises an Fc region (e.g., scFv-Fc), the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the antibody moiety may be made in order to create antibody variants with certain improved properties.

[0245] In some embodiments, the antibody moiety has a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, 73sf-6766980Docket No. 20222-20003.40 FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0246] In some embodiments, the antibody moiety has bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean- Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.). d) Fc region variants

[0247] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody moiety (e.g., scFv-Fc), thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

[0248] In some embodiments, the Fc fragment possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody moiety in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive 74sf-6766980Docket No. 20222-20003.40 assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96®non- radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0249] Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581). In some embodiments, the Fc fragment comprises a N297A mutation. In some embodiments, the Fc fragment comprises a N297G mutation.

[0250] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No.6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem.9(2): 6591-6604 (2001).)

[0251] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises a L234A mutation and / or a L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising a S228P, F234A, and / or a L235A mutation.

[0252] In some embodiments, the antibody moiety comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues). 75sf-6766980Docket No. 20222-20003.40

[0253] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0254] In some embodiments, the antibody variant comprising a variant Fc region comprising one or more amino acid substitutions which alters half-life and / or changes binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which alters binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues, e.g., a modification at position 250 (e.g. , E or Q); 250 and 428 (e.g. , L or F); 252 (e.g. , UY / F / W or T), 254 (e.g. , S or T), and 256 (e.g. , S / R / Q / E / D or T); or a modification at position 428 and / or 433 (e.g. , H / L / R / S / P / Q or K) and / or 434 {e.g., H / F or Y); or a modification at position 250 and / or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 concerning other examples of Fc region variants. e) Cysteine engineered antibody variants

[0255] In some embodiments, it may be desirable to create cysteine engineered antibody moieties, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, 76sf-6766980Docket No. 20222-20003.40 as described further herein. In some embodiments, any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibody moieties may be generated as described, e.g., in U.S. Patent No. 7,521,541. f) Antibody derivatives

[0256] In some embodiments, the antibody moiety (or domain) described herein may be further modified to comprise additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in diagnosis under defined conditions, etc.

[0257] In some embodiments, the antibody moiety (or domain) may be further modified to comprise one or more biologically active protein, polypeptides or fragments thereof. “Bioactive” or “biologically active”, as used herein interchangeably, means showing biological activity in the body to carry out a specific function. For example, it may mean the combination with a particular biomolecule such as protein, DNA, etc., and then promotion or inhibition of the activity of such biomolecule. In some embodiments, the bioactive protein or fragments thereof include proteins and polypeptides that are administered to patients as the active drug substance 77sf-6766980Docket No. 20222-20003.40 for prevention of or treatment of a disease or condition, as well as proteins and polypeptides that are used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, as well as proteins and polypeptides that are administered to a patient to prevent a disease such as a vaccine. Multispecific anti-CD3 constructs

[0258] The disclosure provides anti-CD3 constructs and their shared features thereof. In some embodiments, the anti-CD3 constructs are multispecific antigen-binding molecules (e.g., bispecific CD3 x TAA, trispecific CD3 x TAA1 x TAA2) comprising a first antigen-binding domain that binds CD3, such any one of the acidic pH-selective anti-CD3 antibody domains described herein, and a second antigen-binding domain that binds a second antigen other than CD3. The second antigen can be a disease antigen (i.e., a disease-associated target molecule) such as a tumor associated antigen.

[0259] In some embodiments, the multispecific anti-CD3 constructs bind one or more T cells that express human or cynomolgus monkey CD3E through its anti-CD3 binding domain. In another aspect, the multispecific anti-CD3 constructs bind one or more second-antigen-positive cells that express a second antigen through the second antigen-binding domain.

[0260] In another aspect the disclosure provides a multispecific anti-CD3 construct comprising: i) a first antigen-binding domain that binds to a T cell (e.g., effector T cells) with an EC50 value of greater than about 1 nM but less than about 100 nM, and ii) a second antigen- binding domain that binds a disease antigen-positive cell (e.g., cancer cell) with an EC50 value of greater than about 10 pM but less than about 50 nM, wherein such EC50binding values are measured in a flow cytometric binding assay with an acidic pH of about 6.0-6.6.

[0261] In some embodiments, the multispecific anti-CD3 construct comprises a first antigen- binding domain that is capable of specifically binding each of human CD3 and cynomolgus CD3 with an acidic pH-selective affinity KD that is greater than about 0.1 nM and less than about 50 nM in an assay condition of about pH 6.0-6.6, that is otherwise greater than about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, or greater than about 200 nM in an assay condition of about pH 7.4, wherein such affinity KD values are measured via a surface plasma resonance (e.g., Biacore SPR) or a biolayer interferometry method (e.g., Octet BLI or Gator BLI). 78sf-6766980Docket No. 20222-20003.40

[0262] In some embodiments, the multispecific anti-CD3 constructs, upon contacting one or more T cells (e.g., cytotoxic T cells) and one or more disease-antigen-positive cells, induce T cell-dependent cytotoxicity towards the contacted disease-antigen-positive cells, showing an PA30 or EC50 value of less than about 50 pM, less than about 100 pM, less than about 200 pM , or less than about 500 pM, as measured by using in vitro T cell-dependent cytotoxicity (TDCC) assays under conditions of about pH 6.6 or 6.7 (e.g., pH 6.5, 6.6, 6.7 or 6.8). In some embodiments, the disease-antigen-positive cells are disease associated cells. In some embodiments, disease associated cells are tumor cells that express a tumor associated antigen recognized by the said multispecific anti-CD3 construct. In some embodiments, the disease associated cells are non-tumoral cells such as an autoimmunity-relevant disease cell.

[0263] In some embodiments, the multispecific anti-CD3 constructs, upon contacting one or more T cells and one or more disease-antigen-positive cells, induce the secretion of cytokines (e.g., one, two, three or all of IFNγ, IL-6, TNFα, IL-2, IL-10) with an EC50 value of less than about 50 pM, less than about 100 pM, less than about 200 pM or less than about 500 pM as measured in in vitro cell-based assays at about pH 6.6 or 6.7 (e.g. pH 6.5, 6.6, 6.7, or 6.8). In some embodiments, the disease-antigen-positive cells are disease associated cells. In some embodiments, the disease associated cells are tumor cells that express the tumor associated antigen recognized by the said multispecific anti-CD3 construct. In some embodiments, the disease associated cells are non-tumoral cells such as an autoimmunity-relevant disease cell.

[0264] In general, the disease associated cells such as cancer cell lines used for TDCC and cell-based cytokine induction assays can be procured from a cell repository or a commercial source (e.g., American Cell Type Collection, ATCC). Exemplary disease relevant cells include commonly used tumor cell lines, e.g., MDA-MB-468 cells (triple negative breast cancer line), HPAF-II cells (pancreatic cancer line), SKOV-3 (SKOV-3) cells (ovarian cancer line), Colo 205 cells (colorectal cancer line), BT474 cells (breast cancer line), SKBR3 cells (breast cancer line), MDA-MB-231 cells (breast cancer line), NCI-N87 cells (gastric cancer line), ESO26 cells (esophageal cancer line), Calu3 cells (non-small cell lung cancer line), HCC70 (breast cancer line), A549 (lung cancer line), NCI-H292 (lung cancer line), DU-145 cells (prostate cancer line), LNCaP cells (prostate cancer line), PC-3 cells (prostate cancer line), HCT116 cells (colorectal cancer line), MCF7 cells (breast cancer line) and HepG2 cells (liver cancer line). 79sf-6766980Docket No. 20222-20003.40

[0265] In some embodiments, the multispecific anti-CD3 constructs comprise an anti-CD3 antigen-binding domain and a second antigen-binding domain that recognize a second antigen other than CD3. In some embodiments, the second antigen is a tumor associated antigen. In some embodiments, the second antigen is an autoimmune disease target antigen. In some embodiments, the second antigen is an inflammatory disease target antigen.

[0266] In some embodiments, the multispecific anti-CD3 constructs comprise a) a first antigen-binding domain that is based on any one of the acidic pH-biased anti-CD3 antibody domains described in this disclosure; b) a second antigen-binding domain that specifically recognizes a second antigen (e.g., a tumor associated antigen); and c) a third antigen-binding domain that recognizes a third antigen (e.g., a tumor associated antigen or a non-tumor associated antigen). In some embodiments, the third antigen recognized by the third antigen- binding moiety is a tumor associated antigen that is the same as or different from the foregoing second antigen.

[0267] In some embodiments, the acidic pH-dependent anti-CD3 antibody domain (a first antigen-binding domain), the second antigen-binding domain, and the optional third antigen- binding domain are assembled into an integral multispecific anti-CD3 construct via one or more linkers such as any one of the linkers described herein with any operable form that allows proper functionality and manufacturability of the said multispecific anti-CD3 construct.

[0268] Exemplary non-limiting molecular configurations (i.e., molecular formats) of the multispecific anti-CD3 constructs are illustrated in Figure 1 through Figure 3.

[0269] In some embodiments, the multispecific anti-CD3 construct is a bispecific CD3xTAA T cell engager that comprises an anti-TAA moiety and an acidic pH-selective anti-CD3 domain described in this disclosure. The anti-TAA moiety is an antigen-binding domain that binds a tumor associated antigen (TAA), exemplified by any one of the TAAs selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSA, PSMA, AFP, STEAP1, STEAP2, STEAP3, STEAP4, STEAP1B, BCMA, GPRC5D, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1 (CD227), MUC16 (CA125), MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, FAP, CTHRC1, Nectin-4, B7- H3(CD276), B7-H4, SLC34A2 (NaPi2b), AXL, P-Cadherin, Mer-TK, CDCP1, CD25, CD33, CD38, CD46 (MCP), CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205 (Ly75), CD228 (SEMF), CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, PD- 80sf-6766980Docket No. 20222-20003.40 L1, ROR1, ROR2, ADAM17, FOLRl (FRα), Globo H, IGF-1R, Integrin beta-6, CDH3, CDH17, PTK7 (CCK4), CAIX (CA9), EphA2, 5T4, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6 (LIV1A), TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, FcRH5, Fibronectin, GUCY2C, CDCP1, CDH3, CDH17, HLA-G, peptide-HLA-E, Tn glycan and CDH6.

[0270] In some embodiments, the multispecific anti-CD3 construct is a trispecific CD3xTAA T cell engager that comprises two anti-TAA antigen-binding domains and an acidic pH- selective anti-CD3 domain described in this disclosure. Each of the two anti-TAA binding domains can binds a tumor associated antigen; such two TAAs are exemplified by any one of the TAAs selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSA, PSMA, AFP, STEAP1, STEAP2, STEAP3, STEAP4, STEAP1B, BCMA, GPRC5D, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1 (CD227), MUC16 (CA125), MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, FAP, CTHRC1, CDH3, CDH17, Nectin-4, B7-H3(CD276), B7-H4, SLC34A2 (NaPi2b), AXL, P-Cadherin, Mer-TK, CDCP1, CD25, CD33, CD38, CD46 (MCP), CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205 (Ly75), CD228 (SEMF), CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, PD-L1, ROR1, ROR2, ADAM17, FOLRl (FRα), Globo H, IGF-1R, Integrin beta-6, GUCY2C, (CCK4), CAIX (CA9), EphA2, 5T4, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6 (LIV1A), TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, FcRH5, Fibronectin, HLA-G, peptide-HLA-E, Tn glycan and CDH6.

[0271] The functional properties of a multispecific anti-CD3 construct (e.g., anti-CD3 based T cell engagers) are generally multifaceted. In one aspect, one kind of anti-CD3 based TCE function is the ability of activating T cells (e.g., CD4 and CD8 T cells, NKT cells), which can manifest as an altered level of cell surface markers (CD69, CD25, CD134, CD137, CD62L, PD-1, LAG3, among others).

[0272] In another aspect, the function of anti-CD3 based T cell engagers can be indicated by the elevated release of certain cytokines including chemokines (e.g., IL-2, IFNγ, TNFα, IL-6, IL-10, MCP-1, IL-1, IL-8, CXCL10, IL-12, IL-15, and IFNα) and by the induction of some apoptosis factors (e.g., death receptor ligands such as Fas ligands and TRAIL, which can directly contribute to target-cell killing). The induced cytokines by the TCEs are 81sf-6766980Docket No. 20222-20003.40 pharmacologically relevant to disease (e.g., cancer) treatment because the secreted cytokines can directly promote T cell expansion and indirectly effect proximal immune cells (part of “bystander effects”) to enhance anti-cancer responses, which together can help fight the cancer cells more efficiently. On the other hand, the induced cytokines may trigger harmful adverse effects such as cytokine release syndromes (CRS), vascular leakage, and immune effector cell- associated neurotoxicity syndrome (ICANS). In yet another aspect, the TCEs can elicit the T cell dependent cytotoxicity (“TDCC”) (e.g., cytotoxic T cells dependent) to directly engage and kill a target antigen-positive cell which can be disease cells. TDCC activity can also be elicited against a non-disease cell (e.g., healthy cell) expressing the target antigen molecule, leading to the so-called “on-target off-site” toxicity. On-target off-site toxicity is associated with unfavorable clinical adverse events and patient intolerability. Thus, it has been a great challenge to discover and develop next-generation TCEs that can optimally engage T cells to kill or inhibit disease cells in a potent yet safe manner, in ways that the innovative TCEs will have substantially reduced or minimal toxicities that can be practically tested not only in cell- based assays and rodent-based animal models, but also in a relevant non-human primate species (e.g., cynomolgus monkey). Previously published acidic pH-selective anti-CD3 antibodies are largely suboptimal, which lack adequate anti-tumor efficacy, show suboptimal functional pH- selectivity, and / or lack the cross-reactivity to monkey CD3 required for practical NHP toxicity / safety evaluations (see the U.S. patent documents US63 / 212,024, US63 / 305,588, US62 / 684,818, US62 / 860,092, US11851499 B2; US63 / 109, 105; US63 / 346, 795, US63 / 431, 611, US63 / 496,657; US63 / 858,968; and Frey et al, Mabs, vol. 16, No. 1, 2322562 (2024)).

[0273] In some embodiments, upon contacting a CD3E, a CD3E-containing complex, one or more T cells (i.e., human T cells), the innovative acidic pH-biased anti-CD3 variants or multispecific anti-CD3 constructs described herein promote a stronger binding affinity in conditions with a pH of about 6.5-6.8 (such as pH 6.5 or 6.6), that is at least 3 times that in conditions with a pH of about 7.3 to about 7.5 (such as pH 7.4), correspond to an affinity based functional pH-selectivity index (affinity FPSI) of at least 3 (i.e., at least 3 folds).

[0274] In some embodiments, upon contacting one or more T cells and one or more disease cells (e.g., TAA-positive cells), the innovative acidic pH-biased anti-CD3 constructs described herein promote a robust TDCC activity of the T cells (e.g., cytotoxic T cells) against the disease cells in conditions with a pH of about 6.5 to about 6.8, that is much stronger than the TDCC 82sf-6766980Docket No. 20222-20003.40 activity in conditions with a pH of about 7.3 to about 7.5. For example, the TDCC EC50 values under conditions of about pH 7.4 can be at least about 10 folds, about 12 folds, about 15 folds, about 20 folds, about 30 folds, about 40 folds, at least about 50 folds, about 60 folds, about 70 folds, about 80 folds, about 90 folds, or at least about 100 folds as that under conditions of about pH 6.6 or 6.7, wherein the EC50 ratios (the EC50 at about pH 7.4 over the EC50 at about pH 6.6) can be used to define a TDCC-based functional pH-selectivity index (i.e., “TDCC FPSI”). In contrast, a non-pH-selective CD3xTAA TCE will have a much smaller TDCC FPSI that is generally smaller than 3 folds, smaller than 2 folds or smaller than 1-fold, whereas a weakly pH-selective CD3xTAA TCE may be featured by a TDCC FPSI of about 3 to about 9 folds, or about 4 to about 7 folds.

[0275] In some embodiments, upon contact with one or more T cells and one or more disease- antigen-positive cells (e.g., TAA-positive cells), the anti-CD3 construct described herein induces robust cytokine release in conditions with a pH of about 6.5 to about 6.8, such cytokine induction is much stronger than that in conditions with a pH of about 7.3 to about 7.5. For example, the cytokine-induction EC50 under conditions of about pH 7.4 can be at least about 10 folds, about 12 folds, about 15 folds, about 20 folds, about 30 folds, about 40 folds, at least about 50 folds, about 60 folds, about 70 folds, about 80 folds, about 90 folds, or at least about 100 folds as that under conditions of about pH 6.6, wherein the EC50 fold difference reflects a cytokine-induction based functional pH-selectivity index (“cytokine-release FPSI”). In contrast, a non-pH-selective CD3xTAA TCE will have a much smaller cytokine-induction FPSI that is generally smaller than 3 folds, smaller than 2 folds or smaller than 1-fold, whereas a weakly pH-selective CD3xTAA TCE may be featured by a cytokine-induction FPSI of about 3 to about 9 folds, or about 4 to about 7 folds.

[0276] In some embodiments, the multispecific CD3xTAA construct comprising an acidic pH-selective anti-CD3 domain (h10.v63) exhibits a favorable acidic pH-biased TDCC property, as shown by the exemplary h10.v63 x h101-1.v1 TCE (Fig. 10). In this TDCC assay, the pH- selective h10.v63 xh101-1.v1 TCE targeting TROP-2 showed a relatively robust TDCC activity (EC50: 0.19 nM) under tumor-relevant pH 6.7 conditions, whereas it exhibited considerably weaker TDCC activity (EC50: 18 nM) under physiologically relevant pH 7.4 conditions. This discrepancy resulted in a TDCC FPSI (as defined in the preceding two paragraphs) of about 95-fold. The TROP-2-positive breast cancer cell line, MDA-MB-468, 83sf-6766980Docket No. 20222-20003.40 served as a model of the disease target cell. In contrast, the control TCE (h10.wt x h101-1.v1), which incorporates the same anti-TROP-2, displayed relatively stronger TDCC potency (EC50 nM) under physiologically relevant pH 7.4 in comparison to tumor-relevant pH 6.6 conditions, leading to a TDCC FPSI of about 0.04-fold.

[0277] In another aspect, as displayed in Fig.11, the exemplary CD3xTAA TCE also exhibits a favorable acidic pH-biased cytokine-release property. In this cytokine-secretion assay, the pH-selective h10.v63xh101-1.v1 TCE further showed a much lower cytokine release activity under physiologically relevant pH 7.4 conditions (EC50: >200nM for each of IL-2 and TNFα), whereas it induced considerably higher cytokine release under tumor-relevant pH 6.7 conditions (EC50: 0.2 nM for IL-2, and 1.2 nM for TNFα). This discrepancy resulted in a cytokine-release FPSI of >1000-fold for IL-2 and 166-fold for TNFα respectively. The TROP- 2-positive breast cancer cell line, MDA-MB-468, served as a model of the disease target cell. In contrast, the control TCE (h10.wt x h101-1.v1), which comprises the same anti-TROP-2, displays relatively stronger cytokine-triggering potency under both physiologically relevant pH 7.4 and tumor-relevant pH 6.6 conditions, showing a much lower cytokine-release FPSI of approximately 0.15-fold for IL-2 and 1.5-fold for TNFα respectively.

[0278] In yet another aspect of cytokine-induction activity, as displayed in Figs. 12 and 13, the pH-selective h10.v63xh101-1.v1 TCE further showed a similarly low IFNγ, low IL-6 and low IL-10 release activity under physiologically relevant pH 7.4 conditions, but a considerably higher release of each of IFNγ, IL-6 and IL-10 under tumor-relevant pH 6.7 conditions. The activity differences resulted in a FPSI of >100-fold (IL-2 release), 250-fold (TNFα release), and >100-fold (IL-10 release), respectively. In contrast, the non-pH selective control TCE (h10.wt x h101-1.v1) displayed relatively stronger cytokine-induction activity under both physiologically relevant pH 7.4 and tumor-relevant pH 6.6 conditions, showing a small FPSI of about 1.5-fold for IFNγ, about 1-fold for IL-6, and about 2-fold for IL-10, respectively.

[0279] The observed acidic pH-selective potent activity and favorable FPSI characteristics of the disclosed anti-CD3 constructs are not limited to tumor target (TAA), tumor cell line, or human donor PBMCs, which are the three key components of TDCC or cytokine induction assays for TCEs.

[0280] As shown in Figs.14 and 15, the acidic pH-selective anti-CD3 clones (h10.v63 and a homologous h10.v52) were utilized to pair with an anti-EpCAM antibody domain (VHH16.v2- 84sf-6766980Docket No. 20222-20003.40 1), leading to two acidic pH-selective CD3xTAA TCEs (h10.v63xVHH16.v2-1 and h10.v52 xVHH16.v2-1) that exhibited both potent acidic-pH-biased TDCC and favorably increased FPSI, towards either exemplary breast cancer cell line MDA-MB-468 (Fig.14A) or exemplary colon cancer cell line HT-29 (Fig. 15A). In contrast, the non-pH selective CD3xTAA TCEs (h10.wt xVHH16.v2-1 and h10.v65xVHH16.v2-1) displayed non-pH-selective potent TDCC and a poor FPSI towards either of the target cancer cells.

[0281] As shown in Figs. 16 and 17, the acidic pH-selective anti-CD3 clones (h10.v63 and h10.v52) were utilized to fuse with various anti-TROP-2 antibody domains (h101-1.v6b or h101-1.v12), leading to three acidic pH-selective CD3xTROP-2 TCEs (h10.v63xh101-1.v6b, h10.v63xh101-1.v12, and h10.v52xh101-1.v12). Each of the three TCEs exhibited potent, acidic pH-biased TDCC activity as well as favorable TDCC-based FPSI, towards the exemplary ovarian cancer cell line SKOV-3 (Fig. 16A) and the exemplary pancreatic cancer cell line HPAF-II (Fig. 17A).

[0282] As shown in Figs. 18 and 19, the three acidic pH-selective three acidic pH-selective CD3xTROP-2 TCEs (h10.v63xh101-1.v6b, h10.v63xh101-1.v12, and h10.v52xh101-1.v12) were assessed in cell-based cytokine-induction assays. Each of the three TCEs exhibited acidic pH-biased cytokine induction activity as well as favorable high cytokine-release FPSIs, with respect to IFNγ and IL-2 release (Fig. 18A) and TNFα and IL-6 release (Fig. 19A)

[0283] In some embodiments, the acidic pH-selective CD3xTAA (e.g., h10.v63xh101-1.v1, h10.v63xh101-1.v6b) were subject to the TDCC and cytokine release assays using multiple sources of different human donor PBMCs respectively. The result showed consistent acidic- pH-biased TDCC and favorable high FPSIs of >10 folds, in contrast to the non-pH-selective TDCC and unfavorable low FPSIs of <2 folds, as observed with the non-pH selective control TCEs (e.g., h10.wt x h101-1.v1) (data not shown).

[0284] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v80) was used to fuse with an anti-EGFR antigen-binding domain (h103-1.v1) to generate an acidic pH- selective CD3xEGFR TCE (h10.v80 x h103-1.v1) comprising a so-called “2+1” molecular format comprising one h10.v80 moiety (according to the variable sequences set forth in SEQ ID NOs: 132 and 152), two identical copies of h103-1.v1 (comprising the sequence of SEQ ID NO: 201), and a conventional Knobs-in-Holes IgG1 Fc domain. As exhibited in Fig. 20, the h10.v80 x h103-1.v1 TCE showed a potent TDCC at about pH 6.7 assay conditions (EC50: 85sf-6766980Docket No. 20222-20003.40 ~35 pM), and a weak TDCC at about pH 7.4 assay conditions (EC50: >100,000 pM) towards an EGFRpositivetarget cell line (SKOV-3), reflecting a strong potency in tumor-relevant pH condition and an excellent safety margin with a FPSI of over 2,800 folds.

[0285] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v80) can be used to fuse with the anti-TROP-2 antigen-binding domain (h101-1.v1) to generate an acidic pH-selective CD3xTROP-2 TCE (h10.v80 x h101-1.v1) comprising a regular “1+1” trans- format comprising one h10.v80 moiety, one h101-1.v1 moiety, and a Knobs-in-Holes IgG1 Fc domain. As exhibited in Figs. 21A, the h10.v80 x h101-1.v1 TCE showed a potent TDCC at about pH 6.7 assay conditions (EC50: 0.1 nM), but a weak TDCC at about pH 7.4 assay conditions (EC50: 1.8 nM) towards a TROP-2positivetarget cell line (SKOV-3), reflecting a favorable TDCC FPSI of 18 folds.

[0286] In contrast, as shown in Fig. 21B, under substantially same TDCC assay conditions as described in Fig. 21A, the non-pH selective conventional TCE (h10.wt x h101-1.v1) comprising the non-pH selective anti-CD3 (h10.wt) comprising SEQ ID NOs: 190 and 191, and an anti-TAA (TROP-2) binding domain (SEQ ID NO: 196), showed non-pH-selective, comparable TDCC activities under the pH 6.7 versus pH7.4 assays conditions, reflecting a poor safety margin indicated by an unfavorable TDCC FPSI (about 0.2 folds) of conventional TCEs.

[0287] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v84) comprising SEQ ID NOs:133 and 153 was used to fuse with the anti-TROP-2 antigen-binding domain (h101-1.v1) to generate an acidic pH-selective CD3xTROP-2 TCE (h10.v84 x h101-1.v1) comprising the similar molecular configuration as the TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 22A, under substantially same assay conditions described in Fig. 21 the h10.v84 x h101-1.v1 showed a potent TDCC at about pH 6.7 assay conditions (EC50: 0.01 nM), and a weakened TDCC at about pH 7.4 assay conditions (EC50: 0.03 nM), reflecting a strong potency in tumor-relevant pH condition and a favorable safety margin with a FPSI of 3 folds.

[0288] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v154) comprising SEQ ID NOs: 134 and 154, was used to pair with the anti-TROP-2 antigen-binding domain (h101-1.v1) to generate an acidic pH-selective CD3xTROP-2 TCE (h10.v154 x h101- 1.v1) comprising the similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 22B, under substantially same assay conditions described 86sf-6766980Docket No. 20222-20003.40 in Fig. 21 the h10.v154 x h101-1.v1 TCE showed a potent TDCC at about pH 6.7 assay conditions (EC50: 40 pM), and a weak TDCC at about pH 7.4 assay conditions (EC50: 2.04 nM), reflecting a strong potency in tumor-relevant pH condition and an excellent safety margin with a FPSI of 51 folds.

[0289] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v168) comprising SEQ ID NOs: 135 and 155, was used to pair with the anti-TROP-2 antigen-binding domain (h101-1.v1) to generate an acidic pH-selective CD3xTROP-2 TCE (h10.v168 x h101- 1.v1) comprising the similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 23A, under substantially same assay conditions described in Fig. 21 the h10.v168 x h101-1.v1 TCE showed a potent TDCC at about pH 6.7 assay conditions (EC50: 40 pM), and a weak TDCC at about pH 7.4 assay conditions (EC50: 0.3 nM), reflecting a strong potency in tumor-relevant pH condition and an excellent safety margin with a FPSI of about 7.5 folds.

[0290] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v170) comprising SEQ ID NOs: 136 and 156, was used to pair with the anti-TROP-2 antigen-binding domain (h101-1.v1) to generate an acidic pH-selective CD3xTROP-2 TCE (h10.v170 x h101- 1.v1) comprising the similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 23B, under substantially same assay conditions described in Fig. 21 the h10.v170 x h101-1.v1 TCE showed a potent TDCC at about pH 6.7 assay conditions (EC50: 0.05 nM), and a weak TDCC at about pH 7.4 assay conditions (EC50: 0.9 nM), reflecting a strong potency and an excellent safety margin with a FPSI of 19 folds.

[0291] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v176) comprising SEQ ID NOs: 137and 157, was used to pair with the anti-TROP-2 antigen-binding domain (h101-1.v1) to generate an acidic pH-selective CD3xTROP-2 TCE (h10.v176 x h101- 1.v1) comprising the similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 24A, under substantially same assay conditions described in Fig. 21 the h10.v176 x h101-1.v1 TCE showed a potent TDCC at about pH 6.7 assay conditions (EC50: 0.7 nM), and a weak TDCC at about pH 7.4 assay conditions (EC50: 175 nM), reflecting a favorable high FPSI of 250 folds.

[0292] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v190) comprising SEQ ID NOs: 138 and 158, was used to pair with the anti-TROP-2 antigen-binding 87sf-6766980Docket No. 20222-20003.40 domain (h101-1.v1) to generate an acidic pH-selective CD3xTROP-2 TCE (h10.v190 x h101- 1.v1) comprising the similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 24B, under substantially same assay conditions described in Fig. 21 the h10.v190 x h101-1.v1 TCE showed a potent TDCC at about pH 6.7 assay conditions (EC50: 0.1 nM), and a weak TDCC at about pH 7.4 assay conditions (EC50: 2.7 nM), reflecting a strong potency and an excellent safety margin with a FPSI of 27 folds.

[0293] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v84) comprising SEQ ID NOs: 133 and 153, was used to pair with an tumor antigen binding domain (anti-TAA- 1) to generate an acidic pH-selective CD3xTAA-1 TCE (h10.v84 x TAA-1) comprising a similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 41, under substantially same assay conditions described in Fig. 21 the h10.v190 x h101-1.v1 TCE showed a strong TDCC at about pH 6.8 assay conditions (EC50: 45 pM), and a weak TDCC at about pH 7.4 assay conditions (EC50: >1 nM), reflecting a strong potency in tumor-relevant pH condition and an excellent safety margin with a FPSI of > 22 folds.

[0294] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v138) comprising SEQ ID NOs: 372 and 472, was used to pair with the anti-EGFR antigen-binding domain (h103-1.v17) to generate an acidic pH-selective CD3xEGFR TCE (h10.v138 x h103- 1.v17) comprising a similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 42, under substantially same assay conditions described in Fig. 21 the h10.v138 x h101-1.v1 TCE showed a high TDCC at about pH 6.7 assay conditions (EC50: 0.37 nM), and a weak TDCC at about pH 7.4 assay conditions (EC50: 17 nM), reflecting a favorable high FPSI of ~46 folds.

[0295] In some embodiments, the acidic pH-selective anti-CD3 variant (h10.v138) comprising SEQ ID NOs: 372 and 472, was used to pair with the a tumor antigen binding domain (TAA-2) to generate an acidic pH-selective CD3xTAA-2 TCE (h10.v138 x TAA-2) comprising a similar molecular configuration as these TCEs provided in Figs. 16, 17, 18, 19 and 21. As exhibited in Fig. 43, under substantially same assay conditions described in Fig.21 the h10.v138 x h101-1.v1 TCE showed a high TDCC at about pH 6.8 assay conditions (EC50: ~19 pM), and a weak TDCC at about pH 7.4 assay conditions (EC50: > 1 nM), reflecting a 88sf-6766980Docket No. 20222-20003.40 strong potency in tumor-relevant pH condition and an excellent safety margin with a FPSI of ~52 folds.

[0296] The EC50s of the observed acidic pH-selective TDCC activities and the relevant TDCC-based FPSI parameters are summarized in Table 7, with respect to exemplary multispecific anti-CD3 constructs (e.g., CD3 x TAA TCEs), including those that comprise a pH-selective anti-CD3 variant (e.g., h10.v52, h10.v63, h10.v80, h10.v84, h10.v138, h10.v154, 10.v168, h10.v170, h10.v176, or h10.v190), a non-pH-selective anti-CD3 clone (h10.wt), and those comprising other exemplified pH-selective anti-CD3 variants in Table 5 (additional TDCC data unshown). Table 7. The TDCC EC50s and the TDCC-based FPSIs of exemplary CD3xTAA TCEs.89sf-6766980Docket No. 20222-20003.40

[0297] In general, the CD3xTAA constructs comprising an acidic pH-selective anti-CD3 showed a TDCC-based functional pH-selectivity index of at least 3.0, at least 7.5, or at least 10, regardless of the choice of anti-TAA binding domains (Table 7), the choice of the TAA- positive target cells (Table 7), or the choice of donor T cell source (data not shown).

[0298] Cytokine induction is another critical functional feature of an multispecific anti-CD3 constructs. In some embodiments, the exemplary acidic pH-selective CD3xTAA TCE (h10.v80 x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig. 25, the TCE showed a weak or minimal cytokine secretion at about pH 7.4 assay conditions , but a significantly higher cytokine release at about pH 6.7 assay conditions, reflecting favorable cytokine- induction FPSIs of >32 folds for IL-2, 12.7 folds for IFNγ, >5.7 folds for TNFα, and >52.6 folds for IL-6, respectively.

[0299] In some embodiments, as a control molecule, the exemplary non-pH-selective CD3xTAA TCE (h10.wt x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig.26, the TCE showed strong cytokine secretions at about pH 7.4 assay conditions, reflecting unfavorable cytokine-induction FPSIs of <0.11-fold for IL-2, 0.3-fold for IFNγ, <0.02-fold for TNFα, and 0.1-fold for IL-6, respectively. The relatively high cytokine release activity in the pH 7.4 conditions is generally a critical indicator of toxicity / safety concerns.

[0300] In some embodiments, the exemplary acidic pH-selective CD3xTAA TCE (h10.v84 x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig.27, under substantially same assay conditions described in Fig. 25 the TCE showed a weak or minimal cytokine secretion at about pH 7.4 assay conditions, but a significantly higher cytokine release at about pH 6.7 assay conditions, reflecting favorable cytokine-induction FPSIs of 17.8 folds for IL-2 and 8.2 folds for TNFα respectively.

[0301] In some embodiments, the exemplary acidic pH-selective CD3xTAA TCE (h10.v154 x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig.28, under substantially same assay conditions described in Fig. 25 the TCE showed a weak or minimal cytokine secretion at about pH 7.4 assay conditions, but a significantly higher cytokine release at about pH 6.7 assay conditions, reflecting favorable cytokine-induction FPSIs of >19.7 folds for IL- 2, >10 folds for IFNγ, >36.8 folds for TNFα, and 14.9 folds for IL-6 respectively. 90sf-6766980Docket No. 20222-20003.40

[0302] In some embodiments, the exemplary acidic pH-selective CD3xTAA TCE (h10.v168 x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig.29, under substantially same assay conditions described in Fig. 25 the TCE showed a weak or minimal cytokine secretion at about pH 7.4 assay conditions, but a significantly higher cytokine release at about pH 6.7 assay conditions, reflecting favorable cytokine-induction FPSIs of >31 folds for IL-2, >11 folds for IFNγ, >35.2 folds for TNFα, and 2.8 folds for IL-6 respectively.

[0303] In some embodiments, the exemplary acidic pH-selective CD3xTAA TCE (h10.v170 x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig.30, under substantially same assay conditions described in Fig. 25 the TCE showed a weak or minimal cytokine secretion at about pH 7.4 assay conditions, but a significantly higher cytokine release at about pH 6.7 assay conditions (EC50: pM), reflecting favorable cytokine-induction FPSIs of >9.6 folds for IL-2, >4.4 folds for IFNγ, >14.8 folds for TNFα, and 72 folds for IL-6 respectively.

[0304] In some embodiments, the exemplary acidic pH-selective CD3xTAA TCE (h10.v176 x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig.31, under substantially same assay conditions described in Fig. 25 the TCE showed a weak or minimal cytokine secretion at about pH 7.4 assay conditions, but a significantly higher cytokine release at about pH 6.7 assay conditions towards a TROP-2positivetarget cell line (SKOV-3), reflecting favorable cytokine-induction FPSIs of >12 folds for IFNγ, and 117 folds for IL-6 respectively.

[0305] In some embodiments, the exemplary acidic pH-selective CD3xTAA TCE (h10.v190 x h101-1.v1) was subject to cytokine release assays. As exhibited in Fig.32, under substantially same assay conditions described in Fig. 25 the TCE showed a weak or minimal cytokine secretion at about pH 7.4 assay conditions, but a significantly higher cytokine release at about pH 6.7 assay conditions, reflecting favorable cytokine-induction FPSIs of >15.7 folds for IL- 2, >4.5 folds for IFNγ, >5.5 folds for TNFα, and 10.6 folds for IL-6, respectively.

[0306] The EC50s of the observed acidic pH-selective cytokine-induction activities and the relevant cytokine-induction FPSI parameters are summarized in Tables 8-10, with respect to exemplary multispecific anti-CD3 constructs (e.g., CD3xTAA TCEs).

[0307] Table 8 shows the IFNγ-secretion EC50s at pH 7.4 versus at pH 6.7 respectively and the relevant FPSIs of listed exemplary CD3xTAA constructs, including those that comprise a pH-selective anti-CD3 variant (h10.v52, h10.v63, h10.v80, h10.v84, h10.v154, 10.v168, h10.v170, h10.v176, or h10.v190), or a non-pH-selective anti-CD3 clone (h10.wt). 91sf-6766980Docket No. 20222-20003.40 Table 8. IFNγ-induction EC50s and the related FPSI profile of exemplary CD3xTAA TCEs.

[0308] Table 9 shows the IL-6-secretion EC50s at pH 7.4 versus at pH 6.7 respectively and the underlying FPSI profile of the listed exemplary CD3xTAA constructs, including those that comprise a pH-selective anti-CD3 variant (h10.v52, h10.v63, h10.v80, h10.v84, h10.v154, 10.v168, h10.v170, h10.v176, or h10.v190), or a non-pH-selective anti-CD3 clone (h10.wt). Table 9. IL6-induction EC50s and the related FPSI profile of exemplary CD3xTAA TCEs.92sf-6766980Docket No. 20222-20003.40

[0309] Table 10 shows the summary of cytokine-secretion FPSI profiles of those exemplary CD3xTAA constructs, including those that comprise one of the disclosed pH-selective anti- CD3 variants (h10.v52, h10.v63, h10.v80, h10.v84, h10.v154, 10.v168, h10.v170, h10.v176, or h10.v190), a non-pH-selective anti-CD3 clone (h10.wt), and those comprising other exemplified pH-selective anti-CD3 variants in Table 5 (additional cytokine data unshown). Table 10. Summary of the cytokine FPSI profiles of exemplary CD3xTAA TCEs.93sf-6766980Docket No. 20222-20003.40Note: cytokine-induction FPSIs were based on assays using SKOV3 by default, or MDA- MB-468 cells (marked by asterisk *)

[0310] In general, the CD3xTAA construct comprising one of the disclosed acidic pH- selective anti-CD3 variants (e.g., h10.v52, h10.v63, h10.v80, h10.v84, h10.v138, h10.v154, 10.v168, h10.v170, h10.v176, or h10.v190) would show at least one cytokine-induction FPSI that is 10-fold or higher, wherein such a cytokine can be selected from the list of IL-2, IL-6, IFNγ, and TNFα. In some embodiments, the at least 10-fold cytokine-induction FPSI is based on IL-6 secretion. In some embodiments, the at least 10-fold cytokine-induction FPSI is based on IFNγ secretion. In some embodiments, the at least 10-fold cytokine-induction FPSI is based on IL-2 secretion. In some embodiments, the at least 10-fold cytokine-induction FPSI is based on TNFα secretion. In some embodiments, at least two cytokines selected from IL-2, IL-6, IFNγ and TNFα would show an FPSI of at least 10 folds receptively for a given CD3xTAA construct comprising an anti-CD3 variant selected from h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261 and h10.v263, and comprising an anti-TAA antigen-binding domain.

[0311] In some embodiments, the CD3 x TAA construct comprising a pH-selective anti-CD3 variant (e.g., h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, or h10.v263) and an anti-TAA antigen-binding domain would show a cytokine-induction FPSI of at least 10 folds for each of at least two cytokines that are selected from IL-2, IL-6, IFNγ, and TNFα. In alternative embodiments, the said at least two cytokines are selected from the list of IL-2, IL- 6, IFNγ, TNFα, and IL-10.

[0312] In some embodiments, the CD3 x TAA construct comprising one of the disclosed acidic pH-selective anti-CD3 variants (h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, 94sf-6766980Docket No. 20222-20003.40 h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, h10.v263) and an anti-TAA antigen-binding domain would exhibit one or more of the following three FPSI threshold criteria: (i) a TDCC-based FPSI of at least 3, (ii) a human CD3 binding affinity based FPSI of at least 3, and / or (iii) a cytokine-induction FPSI of at least 10, wherein the cytokine is selected from the list of IL-2, IL-6, IFNγ, and TNFα of human origin.

[0313] In some embodiments, the CD3xTAA construct comprising one of the disclosed acidic pH-selective anti-CD3 variants (h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, h10.v263) and an anti-TAA antigen-binding domain is featured by having a TDCC- based FPSI of at least 7, having a human CD3 binding affinity-based FPSI of at least 3, and / or having two or more cytokine-induction FPSIs that are both 10 or higher, wherein the two or more cytokines are selected from the list of IL-2, IL-6, IFNγ, and TNFα.

[0314] In some embodiments, the CD3xTAA construct comprising one of the disclosed acidic pH-selective anti-CD3 variants (h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, h10.v263) and a general anti-TAA antigen-binding domain is characterized by (i) a TDCC-based FPSI of at least 10 folds, or (ii) at least one cytokine-induction FPSI of 10 folds or higher, wherein the at least one cytokine is selected from the list of IL-2, IL-6, IFNγ, and TNFα.

[0315] In some embodiments, the CD3xTAA construct comprising one of the disclosed acidic pH-selective anti-CD3 variants (h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, h10.v263) and a general anti-TAA antigen-binding domain is characterized by a 95sf-6766980Docket No. 20222-20003.40 CD3 binding affinity-based FPSI of at least 3 (i.e., at least 3 folds), wherein the CD3 is human CD3E.

[0316] In some embodiments, the CD3xTAA construct comprising one of the disclosed acidic pH-selective anti-CD3 variants (h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, h10.v263) and a general anti-TAA antigen-binding domain is characterized by a CD3 binding affinity-based FPSI of at least about 4 or about 5, wherein the CD3 is human CD3E.

[0317] The observed acidic pH-selective TDCC or cytokine-induction property of the multispecific CD3xTAA TCEs or anti-CD3 constructs comprising any one of the disclosed acidic pH-selective anti-CD3 variants (e.g., any one of h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, or h10.v263) are not limited to the listed individual CD3xTAA TCEs, or to the anti-TAA antigen-binding domain’s valency, or to the IgG1 Fc-based multispecific molecule configuration (such as any configuration illustrated in Figures 1, 2 and 3).

[0318] The acidic pH-selective CD3 binding activity is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VHthat comprises a HC-CDR1 sequence selected from SEQ ID NOs: 1, 22, 43, 49, and 326, a HC-CDR2 sequence selected from SEQ ID NOs: 2, 5, 47, 324, 327, 330, and 333, a HC-CDR3 sequence selected from SEQ ID NOs: 3, 6, 9, 15, 337, 340 and 343, further comprising a VLthat comprises a LC-CDR1 sequence selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99, and 413, a LC-CDR2 sequence selected from SEQ ID NOs: 64, 417 and 420, and a LC-CDR3 sequence corresponding to SEQ ID NO: 65.

[0319] The acidic pH-selective TDCC activity is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VH that comprises a HC-CDR1 sequence selected from SEQ ID NOs: 1, 22, 43, 49, and 326, a HC-CDR2 sequence selected from SEQ ID NOs: 2, 5, 47, 324, 327, 330, and 333, a HC-CDR3 sequence selected from SEQ ID NOs: 3, 6, 9, 15, 96sf-6766980Docket No. 20222-20003.40 337, 340 and 343, further comprising a VLthat comprises a LC-CDR1 sequence selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99, and 413, a LC-CDR2 sequence selected from SEQ ID NOs: 64, 417 and 420, and a LC-CDR3 sequence corresponding to SEQ ID NO: 65.

[0320] The observed acidic pH-selective cytokine induction or release activity is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VH that comprises a HC-CDR1 sequence selected from SEQ ID NOs: 1, 22, 43, 49, and 326, a HC-CDR2 sequence selected from SEQ ID NOs: 2, 5, 47, 324, 327, 330, and 333, a HC-CDR3 sequence selected from SEQ ID NOs: 3, 6, 9, 15, 337, 340 and 343, further comprising a VL that comprises a LC- CDR1 sequence selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99, and 413, a LC-CDR2 sequence selected from SEQ ID NOs: 64, 417 and 420, and a LC-CDR3 sequence corresponding to SEQ ID NO: 65.

[0321] The acidic pH-selective binding activity is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VL that comprises three consensus LC-CDRs corresponding to SEQ ID NOs: 450, 451 and 122, and a VHthat comprises three consensus HC-CDRs corresponding to SEQ ID NOs: 350, 351, and 352.

[0322] The acidic pH-selective TDCC or cytokine-induction property is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VLthat comprises three consensus LC-CDRs corresponding to SEQ ID NOs: 450, 451 and 122, and a VH that comprises three consensus HC-CDRs corresponding to SEQ ID NOs: 350, 351, and 352.

[0323] The acidic pH-selective TDCC or cytokine-induction property is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VLthat comprises three consensus LC-CDRs corresponding to SEQ ID NOs: 450, 451 and 122, and a VH that comprises three consensus HC-CDRs corresponding to SEQ ID NOs: 60, 61 and 62.

[0324] The acidic pH-selective TDCC or cytokine-induction property is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VL that comprises three consensus LC-CDRs corresponding to SEQ ID NOs: 120, 121 and 122, and a VH that comprises three consensus HC-CDRs corresponding to SEQ ID NOs: 350, 351, and 352.

[0325] The acidic pH-selective TDCC or cytokine-induction property is applicable to any multispecific CD3xTAA TCE or anti-CD3 construct comprising a VL that comprises three consensus LC-CDRs corresponding to SEQ ID NOs: 120, 121 and 122, and a VHthat comprises three consensus HC-CDRs corresponding to SEQ ID NOs: 60, 61 and 62. 97sf-6766980Docket No. 20222-20003.40

[0326] In some embodiments, the multispecific anti-CD3 constructs with an acidic pH- selective TDCC or an acidic pH-selective cytokine-induction property comprises one, two or three tumor antigen binding domains that binds to one, two or three tumor antigens respectively.

[0327] In some embodiments, the multispecific anti-CD3 constructs with an acidic pH- selective TDCC or an acidic pH-selective cytokine-induction property comprises two tumor antigen binding domains that bind to two different tumor antigens respectively. In some embodiments, the multispecific anti-CD3 constructs with an acidic pH-selective TDCC or an acidic pH-selective cytokine-induction property comprises two different tumor antigen binding domains that bind to two respective epitopes of one tumor antigen (i.e., bi-paratopic binding).

[0328] In some embodiments, the multispecific anti-CD3 constructs with an acidic pH- selective TDCC or an acidic pH-selective cytokine-induction property comprises one or two tumor antigen binding domains and a binding domain that binds to a T cell surface target such as CD28, CD2, CD8, CD4, TCR receptor alpha subunit, TCR receptor beta subunit, 4-1BB, OX40, PD-1, TIGIT, LAG-3, IL-2R, or IL-7R.

[0329] In some embodiments, the acidic pH-selective TDCC and pH-selective cytokine- induction properties can be generalized for many alternative acidic pH-selective anti-CD3 constructs comprising any one of the acidic pH-selective anti-CD3 monoclonal antibody (Mab) variants disclosed herein and a common anti-TAA antigen-binding domain. These alternative acidic pH-selective anti-CD3 constructs will generally comprise one or more of the following characteristics: (i) one or two anti-TAA binding domains that each have a monovalent affinity KD of about 0.01 nM to about 500 nM towards the intended TAA under a disease-relevant assay condition (such as pH ~6.4-6.9); (ii) a generally acceptable production yield (e.g., showing a favorable titer of about ≥ 0.1 mg / mL in a commonly used transient expression system based on Expi293, ExpiCHO, 293-FT, 293T, CHO, or a derivative or alternative cell- based expression system); and (iii) feasible biophysical properties (e.g., generally acceptable thermostability at about 37-40oC, generally acceptable multi-freeze / thaw-cycle stability, generally acceptable solubility).

[0330] It is understood that an average person with reasonable relevant skills may readily utilize any one of the disclosed acidic pH-selective anti-CD3 variants (e.g., h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, 98sf-6766980Docket No. 20222-20003.40 h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, or h10.v263) to fuse with a generally acceptable or traditional anti-TAA antigen-binding domain of such person’s interest to generate a new acidic pH-selective CD3xTAA construct that targets the chosen TAA (such as any one of the disease associated antigens disclosed in this disclosure).

[0331] It is also understood that an average person with reasonable relevant skills may readily utilize any one of the disclosed acidic pH-selective anti-CD3 variants (e.g., h10.v52, h10.v63, h10.v80, h10.v84, h10.v98, h10.v138, h10.v154, h10.v168, h10.v170, h10.v171, h10.v173, h10.v174, h10.v175, h10.v176, h10.v179, h10.v180, h10.v184, h10.v186, h10.v187, h10.v189, h10.v190, h10.v199, h10.v201, h10.v246, h10.v249, h10.v250, h10.v254, h10.v255, h10.v256, h10.v257, h10.v258, h10.v260, h10.v261, or h10.v263) to fuse with a pH-selective anti-TAA antigen-binding domain, wherein both the anti-CD3 binding domain and the anti- TAA binding domain are acidic pH-selective.

[0332] As used herein and thereafter, the exemplary anti-CD3 or anti-TAA antibodies are formatted in a Fab format unless otherwise specified (e.g., “RS7.v1-scFv” specifies a scFv format of the anti-TROP-2 RS7.v1 clone, so on and so forth). As used herein and thereafter, the anti-CD3 and anti-TAA half antibody can be co-expressed and purified as a heterodimerized multispecific antibody comprising the so-called Knobs-in-Holes (KIH) Fc variant of human IgG1 or IgG4 origin, unless otherwise specified. Notwithstanding the foregoing sentences, the antigen-binding region of an anti-TAA antibody can include many other established or alternative formats without limitations to scFv and Fab; likewise, the heterodimeric Fc (if any) of a multispecific anti-CD3 construct can be replaced by many alternative heterodimerization domains without limitation to a Fc with the Knobs-in-holes mutations.

[0333] T cell-dependent killing of an antigen-positive targe cell involves exquisite steps leading to T cell activation and cell death pathways. As common knowledge known to a person with average skills, the T cell activation status can be analyzed using flow cytometry to examine a few T cell activation markers such as CD69, CD25, CD62L, PD-1, CD134, and CD137. 99sf-6766980Docket No. 20222-20003.40

[0334] In similar assays as described in Figures 10, 14, 16, 21, 41, 42 and 43, the CD3xTAA construct comprising an acidic pH-selective anti-CD3 variants (h10.v52, h10.v63, h10.v80, h10.v84, h10.v138, h10.v154, h10.v168, h10.v170, h10.v176, or h10.v190) and a general anti- TAA antigen-binding domain, triggers acidic pH-selective T cell activation, which is consistent with the acidic pH-selective target-cell killing activity profile as exemplified in these figures. In general, under culture conditions with a pH of about 7.3, 7.4, or 7.5, there is relatively much lower activation of any CD4 or CD8 T cells, whereas the T cell activation is relatively much higher and more evident upon under culture conditions with a pH of about 6.5, 6.6, 6.7, or 6.8 (data not shown).

[0335] It is generally advantageous to have a low level of TDCC triggered by the anti-CD3 TCE upon contacting one or more T cells and one or more antigen-positive target cells under a physiologically relevant condition as compared to a disease-relevant acidic microenvironment. For instance, the preferred level of TDCC triggered by an innovative “safer” TCE under a physiologically relevant condition shall be lower than at least about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of that triggered by one and the same anti-CD3 TCE under a pathologically relevant condition (e.g., an acidic tumor microenvironment-like condition).

[0336] It is also generally advantageous to have a low level of cytokine release triggered by the anti-CD3 TCE upon contacting one or more T cells and one or more antigen-positive target cells under a physiologically relevant condition as compared to a disease-relevant acidic microenvironment. For instance, the preferred level of cytokine release induced by an innovative “safer” anti-CD3 TCE under a physiologically relevant condition shall be lower than at least about 50%, about 40%, about 30%, about 20%, about 10%, or about 5% of that triggered by one and the same anti-CD3 TCE under a pathologically relevant condition (e.g., an acidic tumor microenvironment like condition).

[0337] It is further generally advantageous for an innovative “safer” anti-CD3 TCE to have the favorable TDCC and cytokine-induction properties that are described in the foregoing two paragraphs, which can be indicated by a TDCC-based FPSI of at least 3 folds, at least about 7 folds, or at least about 10 folds, and a cytokine-based FPSI of at least about 8 folds, at least about 10 folds, or at least about 15 folds, wherein the cytokine is selected from the list of IL-2, IFNγ, IL-6, and TNFα, or selected from the list of IL-2, IFNγ, IL-6, TNFα and IL-10. 100sf-6766980Docket No. 20222-20003.40

[0338] It is even further advantageous for an anti-CD3 based TCE to have a comparable cross-reactivity towards monkey CD3 in order to enable the practical use of monkey (such as cynomolgus or rhesus monkeys) as a useful model to assess potential toxicity / safety and pharmacokinetics that are highly relevant to humans, wherein the difference between the binding affinity KD values for human CD3 versus monkey CD3 shall be no more than 4 folds at the pH of about 6.5-6.7, and no more than 4 folds either at the pH of about 7.4.

[0339] The pH-selective anti-CD3 antibody domains (corresponding to the sequences represented by h10.v52, h10.v63, h10.v80, h10.v84, h10.v138, h10.v154, h10.v168, h10.v170, h10.v176, or h10.v190), as well as a conventional, non-pH selective anti-CD3 variant (h10.wt) are generally cross-reactive to cynomolgus CD3 with a similar affinity for human and cynomolgus CD3 (Tables 5 and 6).

[0340] In some embodiments, as shown in Fig. 33, the multispecific anti-CD3 construct exemplified by h10.v63xh101-1.v6b TCE, showed acidic pH-biased TDCC mediated by cynomolgus monkey PBMCs, with a comparable TDCC EC50 and FPSI as those based on human PBMCs, wherein the target cell was a cancer cell line (MDA-MB-468) expressing the intended target antigen (human TROP-2).

[0341] In some embodiments, as shown in Fig. 34, an alternative exemplary multispecific anti-CD3 construct (h10.v80 x h101-1.v6b TCE), also displayed acidic pH-biased TDCC mediated by cynomolgus PBMCs, with a comparable TDCC EC50 and FPSI as those derived from human PBMCs, wherein the target cell was a cancer cell line (HCT-116) engineered to stably express the cynomolgus monkey target antigen (cyno TROP-2). The functional activity on cynomolgus CD3 is not limited to h10.v63 or h10.v80, but rather is a shared characteristic of all the acidic pH-selective anti-CD3 antibody domains provided in this application.

[0342] The acidic pH selectivity of the multispecific anti-CD3 construct comprising an acidic pH-selective anti-CD3 domain is not limited to the “Fab x VHH” molecular format shown in Fig. 1C (e.g., h10.v80 x h101-1.v6b), the “Fab x scFv” molecular format (Fig. 1B) (e.g., h10.v63 x RS7.v1), the “VHH fused N-terminally to the heavy chain of anti-CD3 Fab” (Fig. 1G) (e.g., h10.v80 x h101.1.v6b (2+1)), or the “Fab x Tandem VHH repeat” format (Fig. 1D) (e.g., h10.v63 x VHH16.v2-1). For instance, the molecular format can also be a Fab x mixed VHH combination (Fig. 1E), a VHH fused N-terminally to the light chain of anti-CD3 (Fig. 101sf-6766980Docket No. 20222-20003.40 1F), a scFv (anti-CD3) x VHH (anti-TAA) (Fig. 1H), a scFv (anti-CD3) x Fab (anti-TAA), or a scFv (anti-CD3) – VHH (anti-TAA) single polypeptide (Fig. 3A) (data not shown).

[0343] In general, the following advantageous features are shared among the multispecific anti-CD3 constructs comprising an acidic pH-selective anti-CD3 antibody domain disclosed in this application, in the context of engaging T cells and disease cells for the purpose of eradicating the disease cells while mitigating potential toxicities on healthy cells that invariably express the TAA of interest; such advantageous features include: (1) Acidic pH-selective binding to CD3, wherein the binding is relatively much stronger at about pH 6.5, 6.6, 6.7 or 6.8, and relatively weaker or substantially attenuated at pH 7.4 or about pH 7.4 conditions; (2) Acidic pH-selective T-cell-dependent cytotoxicity, wherein the TDCC activity is relatively potent at about pH 6.5, 6.6, 6.7 or 6.8, but relatively weaker or substantially reduced at pH 7.4 or about pH 7.4 conditions; (3) Acidic pH-selective cytokine-induction property, i.e., triggering a relatively high or moderate level of cytokine release at about pH 6.5, 6.6, 6.7 or 6.8, but a substantially reduced or very low level of cytokine release at pH 7.4 or about pH 7.4 conditions; (4) Cross-reactivity to both human and monkey CD3 (e.g., cyno CD3), wherein the binding affinity and the functional pH-selectivity index profile (at about pH 6.5, 6.6, 6.7 or 6.8 versus at about pH7.4) are each comparable, given human T cells versus monkey T cells; and (5) Excellent anti-tumor efficacy in multiple commonly-practiced in vivo efficacy models (e.g., tumor xenograft models in an immune-deficient mouse strain). For instance, excellent anti-tumor efficacy (achieving a tumor growth inhibition rate of ≥80%) is generally expected for a TCE construct at a dose level of no more than 0.8 or 1 mg / kg for such efficacy models.

[0344] In general, the pH-selective multispecific anti-CD3 construct disclosed in this application comprises an anti-disease-antigen binding moiety. In some embodiments, the disease antigen is a TAA (tumor associated antigen). In some embodiments, the disease antigen is a non-TAA molecule on a non-tumorous cell that can contribute to a disease process. In some embodiments, the non-TAA molecule is a marker on myeloid derived suppressor cell, such as CD33. In some embodiments, the non-TAA molecule is a marker on Treg cells, such 102sf-6766980Docket No. 20222-20003.40 as CD25. In some embodiments, the non-TAA molecule is a virus derived peptide that is presented on infected cells. The said TAAs are exemplified by any target selected from the group consisting of HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, STEAP3, SEAP4, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3 (CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, GUCY2C, CDH3,CDH6, CDH17, (CCK4), CAIX (CA9), EphA2, 5T4, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, FAP, HLA-G, HLA-E, NY-ESO and any of its derivative peptides, gp100 and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS mutant and any of its derivative peptides, and a WT1 mutant and any of its derivative peptides.

[0345] In some embodiments, the pH-selective multispecific anti-CD3 construct disclosed in this application comprises at least one pH-selective anti-CD3 antibody domain, and at least one moiety that recognizes a tumor neoantigen (e.g., KRASG12V-peptide / HLA complex, KRASG12D-peptide / HLA complex, NY-ESO-1-peptide / HLA complex). Additional examples of tumor neoantigens and the cognate TCRs or TCR-mimicry proteins have been described in prior arts by Duan and Ho et al (Antib. Ther., 2021, 4:208-211) and Liu et al (J. Hematol. Oncol., 2019, 12:99), among others.

[0346] In some embodiments, the pH-selective multispecific anti-CD3 construct disclosed in this application comprises an antigen-binding domain that binds the disease antigen wherein the disease antigen is a peptide complexed with a peptide presenting receptor (e.g. HLA-I, HLA-II). In some embodiments, the disease antigen is a disease-specific sugar-containing molecule. In some embodiments, the disease antigen is a carbohydrate or glycolipid moiety. In some embodiments, the disease antigen is an RNA molecule that is presented on a target cell surface.

[0347] The disclosed acidic pH-selective anti-CD3 constructs can be used to target a variety of human diseases. In some embodiments, the disease is an abnormal cell proliferation disorder. 103sf-6766980Docket No. 20222-20003.40 In some embodiments, the abnormal cell proliferation disorder is a solid tumor or a liquid cancer such as myeloma, leukemia and lymphoma. In some embodiments, the disease is an auto-immunity condition. In some embodiments, the disease is a neurological degeneration disease. In some embodiments, the disease is an infectious disease. In some embodiments, the disease is an inflammatory disorder. In some embodiments, the disease is a graft versus host diseases (GvHD). In some embodiments, the disease is a transplant rejection condition.

[0348] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising an acidic pH-selective anti-CD3 antigen-binding domain (see Table 3, Table 4 and Sequence Table for the sequences of these anti-CD3 antibody domains); and an anti-TROP-2 antibody domain that is derived from RS7.v1, h101-1.v1, h101-1.v6b, h101- 1.v12 or an alternative anti-TROP-2 domain. See the Sequence Table for the variable region sequences of these anti-TROP-2 antibodies.

[0349] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VH comprises three CDR-Hs having the amino acid sequences of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, the VH having at least 90% sequence identity as the amino acid sequence of SEQ ID NO.130, and the VL comprises three CDR-Ls having the amino acid sequences of SEQ ID NO.63, SEQ ID NO. 64, and SEQ ID NO. 65, the VL having at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 150; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, 5T4, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, GUCY2C, CDH3, CDH17, GUCY2C, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 104sf-6766980Docket No. 20222-20003.40 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H-RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0350] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VH comprises three CDR-Hs having the amino acid sequences of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, the VHhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO.131, and the VL comprises three CDR-Ls having the amino acid sequences of SEQ ID NO.66, SEQ ID NO. 67, and SEQ ID NO. 68, the VLhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 151 ; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, 5T4, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H- RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0351] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VHcomprises three CDR-Hs having the amino acid sequences of SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, the VHhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO.132, and the VLcomprises three CDR-Ls having the amino acid sequences of SEQ ID NO.69, SEQ ID NO. 105sf-6766980Docket No. 20222-20003.40 70, and SEQ ID NO. 71, the VLhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 152 ; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H- RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0352] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antibody moiety comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VHcomprises three CDR-Hs having the amino acid sequences of SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO. 12, the VH having at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 133, and the VLcomprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 72, SEQ ID NO. 73, and SEQ ID NO. 74, the VL having at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 153; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin 106sf-6766980Docket No. 20222-20003.40 beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, 5T4, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, CDCP1, NKG2D, GD2, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H-RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0353] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VH comprises three CDR-Hs having the amino acid sequences of SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15, the VHhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO.134, and the VL comprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 75, SEQ ID NO. 76, and SEQ ID NO. 77, the VL having at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 154; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H-RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0354] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VH comprises three CDR-Hs 107sf-6766980Docket No. 20222-20003.40 having the amino acid sequences of SEQ ID NO. 16, SEQ ID NO.17, and SEQ ID NO.18, the VH having at least 90% sequence identity as the amino acid sequence of SEQ ID NO.135, and the VL comprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 78, SEQ ID NO. 79, and SEQ ID NO. 80, the VLhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 155; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H-RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0355] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antibody moiety comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VHcomprises three CDR-Hs having the amino acid sequences of SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO. 21, the VHhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 136, and the VL comprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 81, SEQ ID NO. 82, and SEQ ID NO. 83, the VLhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 156; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, 108sf-6766980Docket No. 20222-20003.40 CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H- RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0356] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VHcomprises three CDR-Hs having the amino acid sequences of SEQ ID NO.22, SEQ ID NO.23, and SEQ ID NO.24, the VH having at least 90% sequence identity as the amino acid sequence of SEQ ID NO.137, and the VLcomprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 84, SEQ ID NO. 85, and SEQ ID NO. 86, the VLhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 157; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H- RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides. 109sf-6766980Docket No. 20222-20003.40

[0357] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VH comprises three CDR-Hs having the amino acid sequences of SEQ ID NO.25, SEQ ID NO.26, and SEQ ID NO.27, the VH having at least 90% sequence identity as the amino acid sequence of SEQ ID NO.138, and the VL comprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 87, SEQ ID NO. 88, and SEQ ID NO. 89, the VLhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 158; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1,CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, FAP, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H-RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0358] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VHcomprises three CDR-Hs having the amino acid sequences of SEQ ID NO.31, SEQ ID NO.32, and SEQ ID NO.33, the VH having at least 90% sequence identity as the amino acid sequence of SEQ ID NO.372, and the VLcomprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 93, SEQ ID NO. 94, and SEQ ID NO. 95, the VLhaving at least 90% sequence identity as the amino acid sequence of SEQ ID NO. 472 ; and b) a second antigen-binding domain that specifically binds to a tumor associated antigen that is selected from HER2, HER3, TROP-2, EpCAM, EGFR, 110sf-6766980Docket No. 20222-20003.40 EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3(CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H- RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0359] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VHcomprises three CDR-Hs having the amino acid sequences of SEQ ID NO. 60, SEQ ID NO. 61, and SEQ ID NO. 62, and the VLcomprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 120, SEQ ID NO. 121, and SEQ ID NO. 122; b) a second antigen-binding domain that specifically binds to a tumor associated antigen. In some embodies, the multispecific anti-CD3 construct optionally further comprise a third antigen-binding domain that specifically binds to a tumor- associated antigen that is the same as or different from the TAA recognized by the second antigen-binding domain. The second antigen-binding domain and the optional third antigen- binding domain each bind to a tumor associated antigen selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, KLK2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3 (CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, 111sf-6766980Docket No. 20222-20003.40 PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, CDCP1, CDH17, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, FAP, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H-RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides.

[0360] In some embodiments, the present application provides a multispecific anti-CD3 construct comprising a) an anti-CD3 antigen-binding domain comprising a heavy variable region (VH) and a light chain variable region (VL), wherein the VHcomprises three CDR-Hs having the amino acid sequences of SEQ ID NO.350, SEQ ID NO.351, and SEQ ID NO.352, and the VLcomprises three CDR-Ls having the amino acid sequences of SEQ ID NO. 450, SEQ ID NO. 451, and SEQ ID NO. 122; b) a second antigen-binding domain that binds to a tumor associated antigen. In some embodies, the multispecific anti-CD3 construct further comprise a third antigen-binding domain that binds to a tumor-associated antigen that is the same as or different from the TAA recognized by the second antigen-binding domain. The second antigen-binding domain and the optional third antigen-binding domain each bind to a tumor associated antigen selected from HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSMA, AFP, STEAP1, STEAP2, KLK2, BCMA, FcRH5, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, Nectin-4, B7-H3 (CD276), B7-H4, NaPi2b, AXL, P-Cadherin, CDH6, Mer-TK, CDCP1, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197 (CCR7), CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, ROR1, ROR2, ADAM17, FOLRl, Globo H, IGF-1R, Integrin beta-6, PTK7 (CCK4), CAIX (CA9), EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, CDCP1, CDH17, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142 (TF), SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, 17-A1, NKG2D, GD2, FAP, GUCY2C, CDH3, CDH17, HLA-G, HLA-E, a NY-ESO variant and any of its derivative peptides, a gp100 variant and any of its derivative peptides, a K-RAS variant and any of its derivative peptides, a N-RAS or H-RAS variant and any of its derivative peptides, and a WT1 variant and any of its derivative peptides. 112sf-6766980Docket No. 20222-20003.40

[0361] In some embodiments, the multispecific anti-CD3 construct comprises two heavy chains and two light chains, wherein the first heavy chain and the first light chain pair together and form the anti-CD3 antibody moiety, wherein the second heavy chain and the second light chain pair together and form the second antibody moiety which optionally specifically recognizes a tumor-associated antigen, and wherein the two heavy chains each comprises a Fc domain, and the two Fc domains form a Fc fragment which optionally is a Fc fragment from IgG.

[0362] In some embodiments, the multispecific anti-CD3 construct comprises i) a first polypeptide comprising a light chain, ii) a second polypeptide comprising a heavy chain comprising a first Fc domain, and iii) a third polypeptide comprising a single chain fragment (scFv) and a second Fc domain, wherein the first polypeptide and the second polypeptide pair together and form the anti-CD3 antibody moiety, wherein the scFv specifically recognizes the second antigen which is optionally a tumor-associated antigen, and wherein and the first and the second Fc domains form a Fc fragment which optionally is a Fc fragment from IgG.

[0363] In some embodiments, the multispecific anti-CD3 construct comprises i) a first polypeptide comprising a light chain, ii) a second polypeptide comprising a heavy chain comprising a first Fc domain, and iii) a third polypeptide comprising a single domain antibody (sdAb) and a second Fc domain, wherein the first polypeptide and the second polypeptide pair together and form the anti-CD3 antibody moiety, wherein the sdAb specifically recognizes the second antigen which is optionally a tumor-associated antigen, and wherein the first and the second Fc domains form a Fc fragment which optionally is a Fc fragment from IgG.

[0364] In some embodiments, the multispecific anti-CD3 construct comprises i) a first polypeptide comprising a light chain, ii) a second polypeptide comprising a heavy chain comprising a first Fc domain, and iii) a third polypeptide comprising a single chain fragment (scFv) and a second Fc domain, wherein the first polypeptide and the second polypeptide pair together and form the second antibody moiety that optionally recognizes a tumor-associated antigen, wherein the scFv comprises the anti-CD3 antibody domain, and wherein the first and the second Fc domains form a Fc fragment which optionally is a Fc fragment from IgG.

[0365] In some embodiments, the multispecific anti-CD3 construct comprises a first single chain fragment (scFv) comprising the anti-CD3 antibody moiety and a second scFv that specifically recognizes the second antigen which is optionally a tumor-associated antigen, and 113sf-6766980Docket No. 20222-20003.40 wherein optionally the first scFv and the second scFv are fused via a linker (such as any of the linkers described herein). In some embodiments, the first scFv is fused to the N-terminus of the second scFv. In some embodiments, the first scFv is fused to the C-terminus of the second scFv.

[0366] In some embodiments, the multispecific anti-CD3 construct comprises a single chain fragment (scFv) comprising the anti-CD3 antibody moiety and a single domain antibody (sdAb) that specifically recognizes the second antigen which is optionally a tumor-associated antigen, and wherein optionally the scFv and the sdAb are fused via a linker (such as any of the linkers described herein). In some embodiments, the sdAb is fused to the N-terminus of the scFv. In some embodiments, the sdAb is fused to the C-terminus of the scFv.

[0367] In some embodiments, the multispecific anti-CD3 construct comprises an antigen- binding domain (Fab) comprising the anti-CD3 antibody moiety and a single chain fragment (scFv) that specifically recognizes the second antigen which is optionally a tumor-associated antigen, and wherein optionally the Fab and the scFv are fused via a linker (such as any of the linkers described herein). In some embodiments, the Fab is fused to the N-terminus of the scFv. In some embodiments, the Fab is fused to the C-terminus of the scFv.

[0368] In some embodiments, the multispecific anti-CD3 construct comprises i) a single chain fragment (scFv) comprising the anti-CD3 antibody moiety, ii) a first single domain antibody (sdAb) that specifically recognizes the second antigen which is optionally a tumor- associated antigen, and iii) a second sdAb that specifically recognizes a third antigen which is optionally a second tumor-associated antigen or a human serum albumin, and optionally i) the scFv is fused to the N-terminus of the first sdAb via a first linker, and the first sdAb is fused to the N-terminus of the second sdAb via a second linker, or ii) the scFv is fused to the C-terminus of the first sdAb via a first linker and fused to the N-terminus of the second sdAb via a second linker. The first and / or the second linker can be any of the linkers described herein.

[0369] In some embodiments, the multispecific anti-CD3 construct comprising an acidic pH- selective anti-CD3 domain and an anti-disease-antigen domain (e.g., anti-TAA) may further comprise a third antigen-binding domain that bind and activate an immune-signaling receptor other than CD3 or TCR complex (e.g., CD28, 4-1-BB / CD137, CD134, CD7, IL-2R). In some embodiments, the multispecific anti-CD3 construct comprising an acidic pH-selective anti- CD3 domain and an anti-disease-antigen domain (e.g., anti-TAA) may further comprise a third 114sf-6766980Docket No. 20222-20003.40 antigen-binding domain that bind and inhibit an immune-signaling receptor other than CD3 or TCR (e.g., PD-1, LAG3, TIM-3, TIGIT). In some embodiments, the multispecific anti-CD3 construct comprising an acidic pH-selective anti-CD3 domain and an anti-disease-antigen domain (e.g., anti-TAA) may further comprise a third antigen-binding domain that bind and stimulate an immune-signaling receptor on non-T cells (e.g., NK cells, dendritic cells, myeloid or myeloid-derived cells, monocytes, macrophages). In some embodiments, the multispecific anti-CD3 construct comprising an acidic pH-selective anti-CD3 domain and an anti-disease- antigen domain (e.g., anti-TAA) may further comprise a third antigen-binding domain that bind and inhibit an immune-signaling receptor on non-T cells (e.g., NK cells, dendritic cells, myeloid or myeloid-derived cells, monocytes, macrophages).

[0370] In general, the disclosed multispecific anti-CD3 constructs can be used to treat a relevant disease such as tumors. In some embodiments, the disclosed multispecific anti-CD3 construct is a CD3xTAA TCE that can be used to treat a cancer expressing the TAA. For instance, the disclosed CD3xTROP-2 TCEs capable of killing TROP-2-positive cells in an acidic pH-selective manner can be used to treat TROP-2-positive tumors of human subjects, or to treat an abnormal cell proliferation disorders associated with TROP-2 overexpression.

[0371] The term “cancer” as used herein refers to hematological and solid tissue cancers, which include but are not limited to, carcinoma, sarcoma, melanoma, lymphoma, and leukemia. Exemplary cancers are lung cancer, breast cancer, gastric cancer, pancreatic cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, kidney cancer, gall bladder cancer, head and neck carcinomas, brain tumors, sarcoma, melanoma, liver cancer, renal cancer, prostate cancer, ovarian cancer, acute myeloid leukemia (AML), multiple myeloma (MM), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), non- Hodgkin’s lymphoma, and chronic myeloid leukemia (CML). As used herein, the term “cancer” can be used interchangeably with “tumor”, particularly in conditions where the cancer is a solid tissue malignant tumor.

[0372] Fig. 35 depicts an exemplary multispecific anti-CD3 construct, i.e., CD3xTROP-2 (h10.v63xh101-1.v6b IgG1 (KIH)), which comprises an acidic pH-selective anti-CD3 variant (h10.v63) featuring the VH sequence identified as SEQ. ID NO: 131 and the VL sequence identified as SEQ. ID NO: 151. Notably, the h10.v63xh101-1.v6b IgG1 (KIH) demonstrated remarkable efficacy in tumor eradication, achieving near-complete elimination at a dosage of 115sf-6766980Docket No. 20222-20003.40 0.3 mg / kg in an ovarian cancer model (SKOV-3) implanted in immunodeficient mice (NSG) engrafted with human PBMCs, showing a comparable anti-tumor efficacy as the non-pH selective control h10.wtx h101-1.v1 IgG (KIH).

[0373] In some embodiment, as shown in Fig.36, the provided first exemplary multispecific anti-CD3 construct (h10.v63 x h101-1.v6b IgG1 (KIH)), and the second example (h10.v52xh101-1.v6b IgG1 (KIH)) comprising the acidic pH-selective anti-CD3 variant (h10.v52), showed tumor eradiation at 0.3 mg / kg dosing in a breast cancer model (MDA-MB- 468) in immunodeficient mice (NSG) engrafted with human PBMCs.

[0374] In some embodiment, as exhibited in Fig. 37, the provided exemplary multispecific anti-CD3 construct (h10.v80 x h101-1.v6b (2+1) IgG1 (KIH)), featuring the VH sequence identified as SEQ. ID NO:132 and the VL sequence identified as SEQ. ID NO:152, showed dose-dependent tumor inhibition efficacy on the ovarian cancer model (SKOV-3) in immunodeficient mice (NSG) engrafted with human PBMCs.

[0375] In some embodiment, as exhibited in Fig. 38, the provided exemplary multispecific anti-CD3 construct (h10.v80 x h101-1.v6b (2+1) IgG1 (KIH)), demonstrated dose-dependent tumor inhibition efficacy towards the gastric cancer model (NCI-N87) in immunodeficient mice (NSG) engrafted with human PBMCs.

[0376] In some embodiment, as exhibited in Fig. 39, the provided exemplary multispecific anti-CD3 construct (h10.v80 x h101-1.v6b (2+1) IgG1 (KIH)), displayed dose-dependent tumor inhibition efficacy on the triple negative breast cancer model (HCC70) in immunodeficient mice (NSG) engrafted with human PBMCs.

[0377] In some embodiment, as exhibited in Fig. 39, the provided exemplary multispecific anti-CD3 construct (h10.v80 x h101-1.v6b (2+1) IgG1 (KIH)), displayed dose-dependent tumor inhibition efficacy on the pancreatic cancer model (HPAF-II) in immunodeficient mice (NSG) engrafted with human PBMCs.

[0378] In some embodiments, the anti-CD3 construct (such as a CD3 x TAA construct) described in this disclosure is a multispecific antibody that is expressed in vitro recombinantly and formulated as an off-shelf protein therapeutics. In alternative embodiments, the anti-CD3 construct (such as a CD3 x TAA construct) described in this disclosure is expressed in vivo from engineered cells and formulated as a cell therapeutic and directly administered into human subjects. 116sf-6766980Docket No. 20222-20003.40

[0379] In general, it is expected that anti-CD3 constructs (such as a CD3 x TAA construct) administered in vivo can preferentially link T cells in an acidic environment to a cancer cell for direct killing. Moreover, the secreted cytokines from the activated T cells can presumably activate, promote, or mobilize proximal bystander immune cells to achieve enhanced immune response against the tumor cells. In some embodiments the bystander cells can be T cells such as tumor infiltrating T cells and tissue resident T cells. In some embodiments, the bystander cells can be innate immune cells such as dendritic cells, NK cells, neutrophils, basophils, eosinophils, monocytes, and macrophages. In some embodiments, the bystander cells can be adaptive immune cells such as B cells. On the other hand, in a healthy tissue, will not or will only minimally engage T cells and therefore will not or will barely trigger any killing of or cytokine release towards normal cells that may express a target molecule.

[0380] In some embodiments, the anti-CD3 constructs (e.g., a single-polypeptide CD3 x TAA TCE) expressed from cell therapeutic are in a secreted form expressed and secreted from the said therapeutic cells in vivo. As exemplified in Fig. 4, the engineered human cells such as conventional T cells and NK cells will secrete the anti-CD3 construct in vivo. Under physiological-like microenvironment with a pH of near 7.4, the CD3xTAA TCE presumably will barely or only minimally engage T cells and therefore is largely inactive. Under a tissue microenvironment with an acidic pH of about 6.4 to about 7.0 (e.g. a tumor microenvironment of about pH 6.5 to about pH 6.9) and with the presence of target cells, the CD3xTAA TCE will engage and activate T cells for direct killing of target cells, whereas the killing can be further enhanced indirectly by bystander immune cells that are activated or mobilized by the said activated T cells. In some embodiments, the CD3xTAA TCE-expressing cell therapeutics can comprise an engineered, edited, or derivative form of a variety of human cell types including without limitation to T cells, NK cells, CAR-T cells, TCR-T cells, NKT cells, induced NKT cells, macrophages or macrophage progenitors or progenitor-like cells, stem or stem-like cells, mesenchymal cells, induced progenitor cells, induced pluripotent cells, iPSCs, lineage progenitor cells, tissue progenitor cells and hybrid cells.

[0381] In some embodiments, the anti-CD3 construct as a secreted form can be expressed from one or more genetic elements that are integrated into the genome of anti-CD3 construct- expressing therapeutic cells. In some embodiments, the genetic integration of an anti-CD3 construct-encoding sequence can be done by using a lentiviral vehicle or a retroviral vehicle 117sf-6766980Docket No. 20222-20003.40 that typically results in non-site-specific integration. In some embodiments, the genetic integration of an anti-CD3 construct-encoding sequence can be done by using a site-specific integration approach, for instance, a genomic landing-pad approach that is mediated by a recombinase or integrase such as CRISPR-Cas9 and the like, Bxb1 recombinase and the like, φC31 integrase and the like, Wβ integrase and the like, Zinc Finger Nuclease and the like, or mediated by a transposase (e.g., a Sleeping Beauty or PiggyBac transposase) based approach. In some embodiments, the anti-CD3 construct as a secreted form can be expressed from one or more genetic elements that are introduced into anti-CD3 construct-expressing therapeutic cells as a non-integrated genetic vehicle such as an episomal construct, exosomes, extracellular vesicles (EVs), or a circular nucleic acid molecule.

[0382] In some embodiments, the anti-CD3 construct can be expressed in vivo from an RNA- based therapeutic comprising genetic sequences encoding the said anti-CD3 construct, wherein such an RNA therapeutic is formulated for direct administering into human subjects, wherein the said anti-CD3 construct is expressed as a secreted form in the said subjects for therapeutic uses. In some embodiments, the said RNA is provided in a linear RNA format. In some embodiments, the said RNA is provided in a circular RNA format.

[0383] In some embodiments, the anti-CD3 construct can be expressed in vivo from a gene therapeutic (or genetics medicine) comprising genetic sequences encoding the said anti-CD3 construct, wherein such a gene therapeutic is formulated for direct administering directly administered into human subjects, wherein the is expressed as a secreted form in the said subjects for therapeutic uses. In some embodiments, the gene therapeutic comprises a viral vehicle. In some embodiments, the viral vehicle is engineered or derived from a virus that is exemplified by an adenovirus and an adeno-associated virus (AAV) and / or lentivirus.

[0384] In some embodiments, the anti-CD3 constructs (e.g., the CD3xTAA TCEs) described herein comprise one or more linkers between two moieties (e.g., the anti-CD3 antibody moiety and the half-life extending moiety, the anti-CD3 scFv and the full-length antibody in the bispecific antibodies described above). The length, the degree of flexibility and / or other properties of the linker(s) used in the bispecific antibodies may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more particular antigens or epitopes. For example, longer linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiment, a linker 118sf-6766980Docket No. 20222-20003.40 (such as peptide linker) comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non- cleavable linker. In some embodiments, the linker is a cleavable linker.

[0385] Other linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like. Non-peptide linkers

[0386] Coupling of two moieties may be accomplished by any chemical reaction that will bind the two molecules so long as both components retain their respective activities, e.g., binding to CD3 and a second agent in a bispecific antibody, respectively. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. In some embodiments, the binding is covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents may be useful in coupling protein molecules in this context. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0387] Linkers can be applied in the present application are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). In some embodiments, non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)- 119sf-6766980Docket No. 20222-20003.40 toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0388] The linkers described above contain components that have different attributes, thus may lead to bispecific antibodies with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form antibody fusion protein with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less antibody fusion protein available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone. Peptide linkers

[0389] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996 / 34103.

[0390] The peptide linker can be of any suitable length. In some embodiments, the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 99 or more amino acids long. In some embodiments, the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids long, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids. 120sf-6766980Docket No. 20222-20003.40

[0391] An essential technical feature of such peptide linker is that said peptide linker does not comprise any polymerization activity. The characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall’Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). A particularly preferred amino acid in context of the “peptide linker” is Gly. Furthermore, peptide linkers that also do not promote any secondary structures are preferred. The linkage of the domains to each other can be provided by, e.g., genetic engineering. Methods for preparing fused and operatively linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well-known in the art (e.g. WO 99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y.1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001).

[0392] The peptide linker can be a stable linker, which is not cleavable by proteases, especially by Matrix metalloproteinases (MMPs).

[0393] The linker can also be a flexible linker. Exemplary flexible linkers include glycine polymers (G)n(SEQ ID NO: 180), glycine-serine polymers including, for example, (GS)n(SEQ ID NO: 181) or (GGS)n (SEQ ID NO: 182), (GSGGS)n (SEQ ID NO: 183), (GSGGGS)n (SEQ ID NO: 184), and (GGGS)n (SEQ ID NO: 185), where n is an integer of at least one, as well as glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components of an anti-CD3 construct. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem., 11:173-142 (1992)). The ordinarily skilled artisan will recognize that design of an antibody fusion molecule can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired antibody fusion protein structure.

[0394] In some embodiments, the anti-CD3 antibody domain is formatted in a scFv format that comprising a VH and a VL that are linked together by a linker of sufficient length, 121sf-6766980Docket No. 20222-20003.40 flexibility and proper biophysical property to enable proper molecular folding and functionality of the anti-CD3 scFv. Similarly, a linker can be used to fuse an anti-CD3 antibody domain to an anti-disease-antigen -binding domain, in a way to allow property biophysical property and functionality of the intended multispecific anti-CD3 fusion protein. Further to this embodiment, the linker may comprise, for example, the amino acid sequence of such as (GGGGS)n (SEQ ID NO: 186), wherein n is an integer between 1 and 10, e.g. (GGGGS)2 (SEQ ID NO: 187; hereinafter referred to as “(G4S)2” or “GS2”), or (GGGGS)3(SEQ ID NO: 188; hereinafter referred to as “(G4S)3” or “GS3”). In some embodiments, the linker comprises the amino acid sequence of (GSTSGSGKPGSGEGS)n (SEQ ID NO: 189), wherein n is an integer between 1 and 3. Anti-CD3 fusion molecules

[0395] The anti-CD3 constructs in some embodiments comprise an anti-CD3 antibody moiety and a half-life extending moiety. In some embodiments, the half-life extending moiety is an Fc or Fc-derivative fragment. In some embodiments, the half-life extending moiety is an albumin binding domain (e.g., a human albumin binding antibody fragment). In some embodiments, the half-life extending moiety is a transferrin binding moiety (e.g., a human transferrin binding antibody moiety). In some embodiments, the half-life extending moiety is a charged soluble polypeptide fragment rich in glycine, serine and glutamate, e.g., the so-called XTEN polymer described in the U.S. Patent document US14 / 381,199 (Amunix Pharmaceuticals).

[0396] The term “Fc region,” “Fc domain” or “Fc” refers to a C-terminal non-antigen binding region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native Fc regions and variant Fc regions. In some embodiments, a human IgG heavy chain Fc region extends from Cys226 to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present, without affecting the structure or stability of the Fc region. Unless otherwise specified herein, numbering of amino acid residues in the IgG or Fc region is according to the EU numbering system for antibodies, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. 122sf-6766980Docket No. 20222-20003.40

[0397] In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. In some embodiments, the Fc fragment is selected from the group consisting of Fc fragments from IgG1, IgG2, IgG3, IgG4, and combinations and hybrids thereof.

[0398] In some embodiments, the Fc fragment has a reduced effector function as compared to corresponding wildtype Fc fragment (such as at least about 60%, 70%, 80%, 85%, 90%, or 95% reduced effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC)).

[0399] In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises a L234A mutation and / or a L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising a S228P, F234A, and / or a L235A mutation. In some embodiments, the Fc fragment comprises a N297A mutation. In some embodiments, the Fc fragment comprises a N297G mutation.

[0400] Additional non-limiting Fc examples with reduced effector function and / or altered half-life are taught in previous publications including Liu et al (Antibodies, 9, 64 (2020)) and Saunders (Front. Immunol., 10:1296 (2019)).

[0401] In some embodiments, the anti-CD3 antibody moiety and the half-life extending moiety comprised by an anti-CD3 fusion molecule is linked via a linker (such as any of the linkers described in the “Linkers” section).

[0402] In some embodiments, the anti-CD3 fusion protein further comprises a second agent. In some embodiments, the second agent binds to a tumor associated antigen (such as any one of the tumor agents described herein) or an autoimmunity-related target molecule. Nucleic Acids

[0403] Nucleic acid molecules encoding the monospecific and multispecific anti-CD3 antibodies described herein are also contemplated. In some embodiments, there is provided a nucleic acid (or a set of nucleic acids) encoding a full-length anti-CD3 antibody. In some embodiments, there is provided a nucleic acid (or a set of nucleic acids) encoding an anti-CD3 Fab antibody fragment. In some embodiments, there is provided a nucleic acid (or a set of nucleic acids) encoding an anti-CD3 scFv. In some embodiments, there is provided a nucleic 123sf-6766980Docket No. 20222-20003.40 acid (or a set of nucleic acids) encoding an anti-CD3 Fc fusion protein. In some embodiments, there is provided a nucleic acid (or a set of nucleic acids) encoding a bispecific or trispecific anti-CD3 molecule, or a polypeptide portion thereof. In some embodiments, the nucleic acid (or a set of nucleic acids) encoding the anti-CD3 construct described herein may further comprises a nucleic acid sequence encoding a peptide tag (such as protein purification tag, e.g., a His-tag, an AVI-tag).

[0404] Also contemplated here are isolated host cell comprising an anti-CD3 construct, an isolated nucleic acid encoding the polypeptide components of the anti-CD3 construct, or a vector comprising a nucleic acid encoding the polypeptide components of the anti-CD3 construct described herein.

[0405] The present application also includes variants to these nucleic acid sequences. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the anti-CD3 antibody moieties or multispecific anti-CD3 constructs of the present application under at least moderately stringent hybridization conditions.

[0406] The present invention also provides vectors in which a nucleic acid of the present invention is inserted.

[0407] The nucleic acids of the present invention may also be used for nucleic acid immunization, genetics medicine or gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos.5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In some embodiments, the invention provides a gene therapy vector.

[0408] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal or human virus, and a cosmid. Vecto...

Claims

Docket No. 20222-20003.40 CLAIMS 1. An isolated anti-CD3 construct comprising a CD3 binding domain that comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises three complementarity determining regions HC-CDR1, HC-CDR2, and HC- CDR3, each comprising a sequence as set forth in: 1) SEQ ID NOs: 1, 2, and 3; 2) SEQ ID NOs: 4, 5, and 6; 3) SEQ ID NOs: 7, 8, and 9; 4) SEQ ID NOs: 10, 11, and 12; 5) SEQ ID NOs: 13, 14, and 15; 6) SEQ ID NOs: 16, 17, and 18; 7) SEQ ID NOs: 19, 20, and 21; 8) SEQ ID NOs: 22, 23, and 24; 9) SEQ ID NOs: 25, 26, and 27; 10) SEQ ID NOs: 28, 29, and 30; 11) SEQ ID NOs: 31, 32, and 33; 12) SEQ ID NOs: 34, 35, and 36; 13) SEQ ID NOs: 37, 38, and 39; 14) SEQ ID NOs: 40, 41, and 42; 15) SEQ ID NOs: 43, 44, and 45; 16) SEQ ID NOs: 46, 47, and 48; 17) SEQ ID NOs: 49, 50, and 51; 18) SEQ ID NOs: 52, 53, and 54; 19) SEQ ID NOs: 55, 56, and 57; 20) SEQ ID NOs: 58, 59, and 301; 21) SEQ ID NOs: 302, 303, and 304; 22) SEQ ID NOs: 305, 306, and 307; 23) SEQ ID NOs: 308, 309, and 310; 24) SEQ ID NOs: 311, 312, and 313; 25) SEQ ID NOs: 314, 315, and 316; 26) SEQ ID NOs: 317, 318, and 319; 177sf-6766980Docket No. 20222-20003.40 27) SEQ ID NOs: 320, 321, and 322; 28) SEQ ID NOs: 323, 324, and 325; 29) SEQ ID NOs: 326, 327 and 328; 30) SEQ ID NOs: 329, 330, and 331; 31) SEQ ID NOs: 332, 333, and 334; 32) SEQ ID NOs: 335, 336, and 337; 33) SEQ ID NOs: 338, 339, and 340; 34) SEQ ID NOs: 341, 342, and 343; 35) SEQ ID NOs: 350, 351, and 352; or 36) a variant with up to 5 amino acid substitutions in the HC-CDRs; wherein the VLcomprises three complementarity determining regions LC-CDR1, LC- CDR2, and LC-CDR3, each comprising a sequence as set forth in: 1) SEQ ID NOs: 63, 64 and 65; 2) SEQ ID NOs: 66, 67, and 68; 3) SEQ ID NOs: 69, 70, and 71; 4) SEQ ID NOs: 72, 73, and 74; 5) SEQ ID NOs: 75, 76, and 77; 6) SEQ ID NOs: 78, 79, and 80; 7) SEQ ID NOs: 81, 82, and 83; 8) SEQ ID NOs: 84, 85, and 86; 9) SEQ ID NOs: 87, 88, and 89; 10) SEQ ID NOs: 90, 91, and 92; 11) SEQ ID NOs: 93, 94, and 95; 12) SEQ ID NOs: 96, 97, and 98; 13) SEQ ID NOs: 99, 100, and 101; 14) SEQ ID NOs: 102, 103, and 104; 15) SEQ ID NOs: 105, 106, and 107; 16) SEQ ID NOs: 108, 109, and 110; 17) SEQ ID NOs: 111, 112, and 113; 18) SEQ ID NOs: 114, 115, and 116; 19) SEQ ID NOs: 117, 118, and 119; 178sf-6766980Docket No. 20222-20003.40 20) SEQ ID NOs: 401, 402, and 403; 21) SEQ ID NOs: 404, 405, and 406; 22) SEQ ID NOs: 407, 408 and 409; 23) SEQ ID NOs: 410, 411 and 412; 24) SEQ ID NOs: 413, 414, and 415; 25) SEQ ID NOs: 416, 417, and 418; 26) SEQ ID NOs: 419, 420, and 421; 27) SEQ ID NOs: 422, 423, and 424; 28) SEQ ID NOs: 425, 426, and 427; 29) SEQ ID NOs: 428, 429, and 430; 30) SEQ ID NOs: 431, 432, and 433; 31) SEQ ID NOs: 434, 435, and 436; 32) SEQ ID NOs: 437, 438, and 439; 33) SEQ ID NOs: 440, 441, and 442; 34) SEQ ID NOs: 443, 444, and 445; 35) SEQ ID NOs: 450, 451, and 122; or 36) a variant with up to 5 amino acid substitutions in the LC-CDRs; optionally wherein the anti-CD3 construct, upon contacting one or more T cells, triggers increased binding to and increased functional activity on the T cells at a pH of about 6.5-6.8 in comparison to at a pH of about 7.4, and further optionally wherein the anti-CD3 construct binds to both human and cynomolgus CD3.

2. An isolated anti-CD3 construct comprising an antigen-binding domain that specifically binds to CD3 and that comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VHcomprises an amino acid sequence selected from SEQ ID NOs: 130, 131, 132, 133, 134, 135, 136, 137, 138, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, or a variant comprising at least 90% sequence identity to any of these VHsequences thereof, and wherein the VL comprises an amino acid sequence selected from SEQ ID NOs: 150, 151, 152, 153, 154, 155, 156, 157, 158, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 179sf-6766980Docket No. 20222-20003.40 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, or a variant comprising at least 90% sequence identity to any of any of these VL sequences thereof, optionally wherein the anti-CD3 construct, upon contacting one or more T cells, confers increased binding to and functional activity on the T cells at an acidic pH of about 6.0- 6.7 as compared to at a pH of about 7.4; and further optionally wherein the anti-CD3 construct binds to both human and cynomolgus CD3.

3. The anti-CD3 construct of claims 1 and 2, wherein the CD3 antigen-binding domain is a Fab or a scFv.

4. The anti-CD3 construct of claims 1-3, wherein the construct comprises a half-life extending domain.

5. The anti-CD3 construct of claims 1-4, wherein the construct comprises an Fc or Fc-like domain derived from human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, or the combinations and hybrids thereof.

6. The anti-CD3 construct of claims 1-4, wherein the construct comprises a half-life extending domain that specifically binds to human serum albumin or transferrin.

7. The anti-CD3 construct of claims 1-6, wherein the construct is a multispecific construct that comprises a second antigen-binding domain that specifically binds to a disease antigen.

8. The anti-CD3 construct of claims 1-7, wherein the disease antigen-binding domain comprises a Fab, a scFv, a VHH, a nanobody, a single domain antibody, a Fab’, a F(ab’)2, an Fv, a disulfide stabilized Fv (dsFv), a (dsFv)2, a Fv-Fc fusion, a scFv-Fc fusion, a scFv- Fv fusion, a single chain Fc (scFc), a diabody, a tribody, a tetrabody, an alternative non- antibody scaffold, or a cell-surface receptor fragment.

9. The anti-CD3 construct of claims 1-8, wherein the disease antigen is a tumor associated antigen.

10. The anti-CD3 construct of claims 1-8, wherein the disease antigen is a cell proliferation disorder associated antigen.

11. The anti-CD3 construct of claims 1-8, wherein the disease antigen is an antigen associated with an autoimmunity or inflammatory disease. 180sf-6766980Docket No. 20222-20003.40 12. The anti-CD3 construct of claims 1-11, wherein upon contacting a human CD3, exhibits a monovalent binding affinity at about pH 7.4 with a KD value that is at least 3 times that at about pH 6.5 or 6.

6.

13. The anti-CD3 construct of claims 1-11, wherein upon contacting a cytotoxic T cell and a disease antigen-positive cell, triggers T cell cytotoxicity at about pH 7.4 showing an EC50 at least 7 times that at about pH 6.5 to 6.

8.

14. The anti-CD3 construct of claims 1-13, wherein upon triggering a T cell dependent cytotoxicity (TDCC) on disease antigen-positive cells, shows a TDCC-based functional pH-selectivity index (FPSI) of at least 7.

15. The anti-CD3 construct of claims 1-14, wherein upon contacting a T cell and a disease antigen-positive cell, induces cytokine release at about pH 7.4 showing an EC50 at least 10 times that at about pH 6.5 to 6.8, wherein the cytokine is selected from the list of IL-2, IFNγ, IL-6 and TNFα.

16. The anti-CD3 construct of claims 1-15, wherein upon inducing cytokine release in the presence of T cells and disease antigen-positive cells, shows at least one cytokine- induction FPSI of at least 10, wherein the at least one cytokine is selected from the list of IL-2, IFNγ, IL-6 and TNFα.

17. The anti-CD3 construct of claims 1-16, wherein the construct comprises an antigen- binding domain that specifically binds to an antigen selected from the list of HER2, HER3, TROP-2, EpCAM, EGFR, EGFRvIII, ErbB4, CEA, CEACAM5, c-MET, PSA, PSMA, AFP, STEAP1, STEAP2, STEAP3, STEAP4, KLK2, BCMA, GPRC5D, GPC3, GPC4, ENPP3, CLDN6, CLDN18.2, MSLN, MUC1, MUC16, MUC17, CLEC12A, 5T4, SSTR2, SSTR5, DLL1, DLL3, DLL4, TF-011, FAP, Nectin-4, B7-H3, B7-H4, SLC34A2, AXL, Mer-TK, CDH6, P-Cadherin, CDCP1, CDH3, CDH17, CD25, CD33, CD38, CD46, CD70, CD71, CD73, CD74, CD123, CD166, CD174, CD197, CD205, CD228, CD326, CRIPTO, ED-B, GPR20, GCC, Alpha v beta 6, PD-L1, ROR1, ROR2, ADAM17, FOLR1, Globo H, IGF-1R, Integrin beta-6, PTK7, GUCY2C, CAIX, EphA2, gpA33, IGF1R, PDGFR-α, PDGFR-β, PRLR, TMPRSS2, ULBP2, FGFR2, FRFR3, MCSP, SSEA3, LGR5, CD142, SLC44A4, SLC39A6, TENB2, AGS-16, GPNMB, CYP17A1, NKG2D, GD2, FcRH5, Fibronectin, CTHRC1, HLA-G, HLA-E, Tn glycan, and a mutated variant of NY-ESO, 181sf-6766980Docket No. 20222-20003.40 TP53, BRAF, N-RAS, H-RAS or K-RAS (e.g., K-RASG12D, K-RASG12V), and a tumor neoantigen derived from a cancer-associated mutation.

18. The anti-CD3 construct of claims 1-17, wherein upon triggering the TDCC on disease antigen-positive cells, shows (i) a TDCC-based FPSI of at least 3 and (ii) a cytokine- induction FPSI of at least 10, wherein the cytokine is IL-2, IFNγ, IL-6 and TNFα.

19. The anti-CD3 construct of claims 1-18, wherein the construct comprising at least two antigen-binding domains that specifically binds to a same or two different tumor associated antigens.

20. A pharmaceutical composition comprising the anti-CD3 construct of any one of claims of 1-19, and a pharmaceutically acceptable carrier.

21. An isolated nucleic acid encoding the anti-CD3 construct of any one of claims 1-19.

22. A vector comprising the isolated nucleic acid of claim 21.

23. An engineered virus encoding the isolated nucleic acid of claim 22.

24. An isolated cell comprising the isolated nucleic acid of claim 21, or the vector of claim 22.

25. An immunoconjugate comprising the anti-CD3 construct of any one of claims 1-24, linked to a therapeutic agent or a label.

26. A method of treating a disease or condition in an individual, comprising administering to the individual an effective amount of the anti-CD3 construct of any one of claims 1-25, or the pharmaceutical composition of claim 20.

27. The method of claim 26, wherein the disease or condition is a cancer, a cell proliferation disorder, an autoimmune disease, an inflammatory disorder, allergy, graft versus host diseases (GvHD), or a transplant rejection condition.

28. The method of claim 26, wherein the individual is a human.

29. A kit comprising the pharmaceutical composition of claim 20 and an instruction for treating a disease or condition.

30. An isolated polypeptide comprising a CD3 binding domain that comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a HC-CDR1 selected from SEQ ID NOs: 1, 22, 43, 49 and 326, a HC-CDR2 selected from SEQ ID NOs: 2, 5, 47, 324, 327, 330 and 333, and a HC-CDR3 selected from SEQ ID NOs: 3, 6, 9, 15, 337, 340 and 343, or a variant thereof comprising up to five amino acid alterations in the HC-CDRs; 182sf-6766980Docket No. 20222-20003.40 wherein the VLcomprises a LC-CDR1 selected from SEQ ID NOs: 63, 69, 75, 81, 87, 99 and 413, a LC-CDR2 selected from SEQ ID NOs: 64, 417 and 420, and a LC-CDR3 set forth in SEQ ID NO: 65, or a variant thereof comprising up to five amino acid alterations in the LC-CDRs.

31. A pharmaceutical composition comprising the polypeptide of claim 30.

32. An isolated cell expressing the polypeptide of claim 30.

33. An engineered vector or virus encoding the polypeptide of claim 30. 183sf-6766980

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