Proteins containing kallikrein-related peptidase 2 antigen binding domain and uses thereof

Abstract

Embodiments of the present invention provide isolated proteins comprising an antigen binding domain that binds to kallikrein-related peptidase 2 (hK2), including monospecific antibodies and bispecific antibodies. Additional embodiments of the present invention provide polynucleotides encoding hK2-specific proteins, vectors, host cells, and methods of making and using the same.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on June 23, 2020, is named JBI6125USNP1_SL.txt, and is 764,265 bytes in size. Technical Field

[0003] The present invention provides antigen-binding domains that bind to kallikrein-related peptidase 2 (hK2), proteins comprising antigen-binding domains that bind to hK2, polynucleotides encoding the proteins, vectors, host cells, and methods of making and using the same. Background Art

[0004] Prostate cancer is the second most commonly diagnosed cancer and the sixth leading cause of cancer death in men, accounting for 14% (903,500) of all new cancer cases and 6% (258,400) of all cancer deaths in men worldwide. The progression of prostate cancer from diagnosis to death is best categorized into a series of clinical stages based on the extent of disease, hormonal status, and the presence of detectable metastases: localized disease, rising prostate-specific antigen (PSA) levels after radiation therapy or surgery without detectable metastases, and clinical metastases in the non-castrate or castrate stage. Although surgery, radiation, or a combination of these two may be effective for patients with localized disease, a significant proportion of these patients will develop recurrent disease, as evidenced by rising PSA levels, which can lead to the development of metastases, particularly in high-risk groups—conversion to the lethal stage of the disease.

[0005] Androgen depletion therapy (ADT) is a standard of care for patients with metastatic prostate cancer with the following generally predictable outcome: a decline in PSA levels, a period of stabilization in which the tumor does not proliferate, followed by a rise in PSA levels and regrowth to castration-resistant disease. Historically, ADT has been the standard of care for patients with metastatic prostate cancer.

[0006] Kallikrein-related peptidase 2 (hK2, HK2) is a trypsin-like enzyme with androgen receptor (AR)-driven expression specific for prostate tissue and prostate cancer. hK2 is activated by transmembrane serine protease 2 (TMPRSS2) and secreted into the prostate ducts, where it triggers a cascade of cleavage of the extracellular matrix in semen, namely seminal vesicle proteins, to enhance sperm motility. hK2 expression is limited to prostate and prostate cancer tissue, however, it has recently been demonstrated that hK2 can be detected in breast cancer lines and primary patient samples after appropriate activation of the AR-pathway by steroid hormones (US Patent Publication No. 2018 / 0326102). Similar to PSA, when the highly structured tissue of the prostate is damaged after excessive enlargement or malignant transformation, catalytically inactive hK2 is retrogradely released into the blood.

[0007] There is a need for next generation hK2 binding domains for therapeutic and diagnostic purposes. Summary of the Invention

[0008] The present disclosure provides an isolated protein comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises:

[0009] or the HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 162 and the LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 163; or the HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 164 and the LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 165; or the HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 166 and the LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 167; NO: 167 LCDR1, LCDR2 and LCDR3 of the VL; or SEQ ID NO: 168 HCDR1, HCDR2 and HCDR3 of the VH and SEQ ID NO: 169 LCDR1, LCDR2 and LCDR3 of the VL; or SEQ ID NO: 204 HCDR1, HCDR2 and HCDR3 of the VH and SEQ ID NO: 205 LCDR1, LCDR2 and LCDR3 of the VL.

[0010] The present disclosure also provides an isolated antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises a heavy chain variable region (VH) of SEQ ID NO: 75 and a light chain variable region (VL) of SEQ ID NO: 74.

[0011] The present disclosure also provides isolated antigen binding domains that bind hK2, comprising certain VH and VL amino acid sequences.

[0012] The present disclosure also provides an isolated multispecific protein comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises heavy chain complementarity determining regions (HCDRs) 1, HCDR2, and HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 137 and light chain complementarity determining regions (LCDRs) 1, LCDR2, and LCDR3 of a light chain variable region (VL) of SEQ ID NO: 138; or HCDRs 1, HCDR2, and HCDR3 of a VH of SEQ ID NO: 137 and LCDRs 1, LCDR2, and LCDR3 of a VL of SEQ ID NO: 138; or HCDRs 1, HCDR2, and HCDR3 of a VH of SEQ ID NO: 162 and LCDRs 1, LCDR2, and LCDR3 of a VL of SEQ ID NO: 163; or HCDRs 1, HCDR2, and HCDR3 of a VH of SEQ ID NO: 164 and LCDRs 1, LCDR2, and LCDR3 of a VL of SEQ ID NO: 165. NO:165 VL's LCDR1, LCDR2 and LCDR3; or SEQ ID NO:166 VH's HCDR1, HCDR2 and HCDR3 and SEQ ID NO:167 VL's LCDR1, LCDR2 and LCDR3; or SEQ ID NO:168 VH's HCDR1, HCDR2 and HCDR3 and SEQ ID NO:169 VL's LCDR1, LCDR2 and LCDR3; or SEQ ID NO:204 VH's HCDR1, HCDR2 and HCDR3 and SEQ ID NO:205 VL's LCDR1, LCDR2 and LCDR3. In a specific embodiment, the present disclosure also provides an isolated multispecific protein comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 162 and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 163.

[0013] The present disclosure also provides an isolated multispecific protein comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises a heavy chain variable region (VH) of SEQ ID NO: 75 and a light chain variable region (VL) of SEQ ID NO: 74.

[0014] The present disclosure also provides isolated multispecific proteins comprising an antigen binding domain that binds hK2, the antigen binding domain comprising certain VH and VL amino acid sequences.

[0015] The present disclosure also provides an isolated chimeric antigen receptor (CAR) comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2).

[0016] The present disclosure also provides an isolated chimeric antigen receptor (CAR), the isolated chimeric antigen receptor comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of a heavy chain variable region (VH) of SEQ ID NO: 137 and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of a light chain variable region (VL) of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 137 and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 138; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 162 and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 163; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 164 and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 165. NO:165 VL's LCDR1, LCDR2 and LCDR3; or SEQ ID NO:166 VH's HCDR1, HCDR2 and HCDR3 and SEQ ID NO:167 VL's LCDR1, LCDR2 and LCDR3; or SEQ ID NO:168 VH's HCDR1, HCDR2 and HCDR3 and SEQ ID NO:169 VL's LCDR1, LCDR2 and LCDR3; or SEQ ID NO:204 VH's HCDR1, HCDR2 and HCDR3 and SEQ ID NO:205 VL's LCDR1, LCDR2 and LCDR3. In a specific embodiment, the present disclosure also provides an isolated chimeric antigen receptor (CAR), which comprises an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises heavy chain complementarity determining regions (HCDR) 1, HCDR2 and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 162 and light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 163.

[0017] The present disclosure also provides an isolated chimeric antigen receptor (CAR), comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises a heavy chain variable region (VH) of SEQ ID NO: 75 and a light chain variable region (VL) of SEQ ID NO: 74.

[0018] The present disclosure also provides an isolated multispecific protein comprising a first antigen binding domain that binds hK2 and a second antigen binding domain that binds a lymphocyte antigen, such as CD3.

[0019] In a specific embodiment, the present disclosure provides an isolated multispecific protein comprising a first antigen binding domain that binds hK2 and a second antigen binding domain that binds a lymphocyte antigen, wherein the isolated multispecific protein is an isolated anti-hK2 / anti-CD3 protein.

[0020] The present disclosure also provides immunoconjugates comprising the isolated antigen-binding domain of the present disclosure that binds hK2.

[0021] The present disclosure also provides immunoconjugates comprising an isolated protein comprising an hK2-binding antigen binding domain of the present disclosure.

[0022] The present disclosure also provides immunoconjugates comprising an isolated multispecific protein comprising an hK2-binding antigen-binding domain of the present disclosure.

[0023] The present disclosure also provides immunoconjugates comprising an isolated CAR comprising an hK2-binding antigen binding domain of the present disclosure.

[0024] The present disclosure also provides pharmaceutical compositions comprising the isolated antigen-binding domain of the present disclosure that binds hK2.

[0025] The present disclosure also provides pharmaceutical compositions comprising an isolated protein comprising the hK2-binding antigen-binding domain of the present disclosure.

[0026] The present disclosure also provides pharmaceutical compositions comprising an isolated multispecific protein comprising an hK2-binding antigen-binding domain of the present disclosure.

[0027] The present disclosure also provides pharmaceutical compositions comprising the isolated multispecific proteins of the present disclosure.

[0028] The present disclosure also provides pharmaceutical compositions comprising an isolated CAR comprising an hK2-binding antigen binding domain of the present disclosure.

[0029] The present disclosure also provides isolated polynucleotides encoding the isolated antigen binding domains of the present disclosure that bind hK2.

[0030] The present disclosure also provides isolated polynucleotides encoding isolated proteins comprising an hK2-binding antigen binding domain of the present disclosure.

[0031] The present disclosure also provides isolated polynucleotides encoding isolated multispecific proteins comprising an antigen binding domain of the present disclosure that binds hK2.

[0032] The present disclosure also provides isolated polynucleotides encoding an isolated CAR comprising an antigen binding domain of the present disclosure that binds hK2.

[0033] The present disclosure also provides a vector comprising the polynucleotide of the present disclosure.

[0034] The present disclosure also provides a host cell comprising the polynucleotide or vector of the present disclosure.

[0035] The present disclosure also provides a method of treating an hK2-expressing cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding domain that binds hK2, a protein comprising an antigen binding domain that binds hK2, a multispecific protein comprising an antigen binding domain that binds hK2, a CAR-T comprising an antigen binding domain that binds hK2, an immunoconjugate of the present disclosure, or a pharmaceutical composition of the present disclosure, for a time sufficient to treat the hK2-expressing cancer.

[0036] The present disclosure also provides a method of reducing the amount of hK2-expressing tumor cells in a subject, the method comprising administering to the subject an antigen binding domain that binds hK2, a protein comprising an antigen binding domain that binds hK2, a multispecific protein comprising an antigen binding domain that binds hK2, a CAR-T comprising an antigen binding domain that binds hK2, an immunoconjugate of the present disclosure, or a pharmaceutical composition of the present disclosure, for a time sufficient to reduce the amount of hK2-expressing tumor cells.

[0037] The present disclosure also provides a method of preventing the establishment of a cancer that expresses hK2 in a subject, the method comprising administering to a subject in need thereof an antigen-binding domain that binds hK2, a protein comprising an antigen-binding domain that binds hK2, a multispecific protein comprising an antigen-binding domain that binds hK2, a CAR-T comprising an antigen-binding domain that binds hK2, an immunoconjugate of the present disclosure, or a pharmaceutical composition of the present disclosure to prevent the establishment of the hK2-expressing cancer in the subject.

[0038] The present disclosure also provides a method of treating a non-cancerous condition in a subject at risk of developing a cancer that expresses hK2, the method comprising administering to a subject in need thereof an antigen binding domain that binds hK2, a protein comprising an antigen binding domain that binds hK2, a multispecific protein comprising an antigen binding domain that binds hK2, a CAR-T comprising an antigen binding domain that binds hK2, an immunoconjugate of the present disclosure, or a pharmaceutical composition of the present disclosure to treat the non-cancerous condition.

[0039] The present disclosure also provides a method of treating prostate cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding domain that binds hK2, a protein comprising an antigen binding domain that binds hK2, a multispecific protein comprising an antigen binding domain that binds hK2, a CAR-T comprising an antigen binding domain that binds hK2, an immunoconjugate of the present disclosure, or a pharmaceutical composition of the present disclosure, for a time sufficient to treat the prostate cancer.

[0040] The present disclosure also provides a method of treating breast cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen binding domain that binds hK2, a protein comprising an antigen binding domain that binds hK2, a multispecific protein comprising an antigen binding domain that binds hK2, a CAR-T comprising an antigen binding domain that binds hK2, an immunoconjugate of the present disclosure, or a pharmaceutical composition of the present disclosure, for a time sufficient to treat the breast cancer.

[0041] The present disclosure also provides a method for detecting prostate cancer or breast cancer in a subject, comprising administering the immunoconjugate of the present disclosure to the subject, and detecting binding of the immunoconjugate to hK2, thereby detecting prostate cancer or breast cancer.

[0042] The present disclosure also provides a kit comprising an antigen-binding domain that binds to hK2, a protein comprising an antigen-binding domain that binds to hK2, a multispecific protein comprising an antigen-binding domain that binds to hK2, a CAR-T comprising an antigen-binding domain that binds to hK2, an immunoconjugate of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0043] The present disclosure also provides anti-idiotypic antibodies that bind to the hK2-binding antigen binding domain of the present disclosure.

[0044] The present disclosure also provides a chimeric antigen receptor (CAR), comprising: an extracellular domain comprising an antigen binding domain that binds hK2; a transmembrane domain; and an intracellular signaling domain, optionally comprising at least one costimulatory domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The foregoing will be apparent from the following more particular description of exemplary embodiments as illustrated in the accompanying drawings. TM: transmembrane.

[0046] Figure 1 Shown is a sequence alignment of the VH domains of mu11B6 (SEQ ID NO: 125), hu11B6 (SEQ ID NO: 5), KL2B357 (SEQ ID NO: 159), KL2B358 (SEQ ID NO: 161), KL2B359 (SEQ ID NO: 139), KL2B360 (SEQ ID NO: 159), HCF3 (SEQ ID NO: 6), and HCG5 (SEQ ID NO: 4).

[0047] Figure 2 Shown is a sequence alignment of the VL domains of mu11B6 (SEQ ID NO: 124), hu11B6 (SEQ ID NO: 2), KL2B357 (SEQ ID NO: 160), KL2B358 (SEQ ID NO: 140), KL2B359 (SEQ ID NO: 140), KL2B360 (SEQ ID NO: 140), LDC6 (SEQ ID NO: 1), and LCB7 (SEQ ID NO: 3).

[0048] Figure 3 The binding epitopes of selected hK2 antibodies mapped onto the hK2 antigen sequence are shown. The figure discloses each sequence as SEQ ID NO: 467.

[0049] Figure 4A and Figure 4B Binding of hybridoma supernatants to primary human T cells is shown. Clone UCHT1 was used as a positive control ( Figure 1 B); Mouse IgG1 isotype (mIgG1) was used as a negative control.

[0050] Figure 5 Binding of anti-CD3 scFv variants expressed in E. coli to CD3 is shown.

[0051] Figure 6An alignment of the VL regions of CD3B815 (SEQ ID NO: 249), CD3W244 (SEQ ID NO: 250), CD3W245 (SEQ ID NO: 251), CD3W246 (SEQ ID NO: 252), CD3W247 (SEQ ID NO: 253), and CD3W248 (SEQ ID NO: 254) is shown.

[0052] Figure 7 Shown are hydrogen-deuterium exchange mass spectra (HDX-MS) of CD3W245 binding to human CD3ε (CD3ε:CD3W245) and OKT3 binding to CD3ε (CD3ε:OKT). The amino acid sequence shown represents residues 2-93 of the 105 residues of the ECD domain of CD3ε and corresponds to residues 1-91 of CD3ε SEQ ID NO:340. Single underlining indicates segments where deuteration levels are reduced by 10%-30% in the presence of the antibody compared to CD3ε alone, while double underlining indicates segments where deuteration levels are reduced by >30%.

[0053] Figure 8A Shown are in vitro target cell cytotoxicity of the KL2BxCD3 bispecific molecule measured by the real-time incuCyte imaging system for quantification of target cell death.

[0054] Figure 8B Shown are in vitro target cell cytotoxicity of the KL2BxCD3 bispecific molecules as measured by a fluorescent caspase 3 / 7 reagent used to measure apoptotic signals resulting from target cell death.

[0055] Figure 9A In vitro T cell activation and proliferation by the KLK2xCD3 bispecific antibody were shown by displaying the frequency of CD25-positive cells at different doses.

[0056] Figure 9B In vitro T cell activation and proliferation by the KLK2xCD3 bispecific antibody are shown by displaying the frequency of cells entering the proliferation gate.

[0057] Figure 10A Shown is in vitro T cell INF-γ release by the KLK2xCD3 bispecific antibody.

[0058] Figure 10B Shown is in vitro T cell TNF-α release by the KLK2xCD3 bispecific antibody.

[0059] Figure 11A schematic diagram of the design of hK2-binding chimeric artificial receptors (CARs) is shown. ScFvs binding to hK2 were cloned in various CARs in either VH-VL or VL-VH orientations.

[0060] Figure 12A and Figure 12B HK2 CAR expression on T cell surface is shown.Primary human T cells were electroporated with the following items: no mRNA (simulation) was used, or 10 μg of hK2scFv CAR or unrelated control CAR mRNA was expressed. After staining with 2 μg / ml biotinylated L protein and streptavidin-conjugated PE (upper figure) or 2 μg / ml biotinylated L protein and streptavidin-conjugated PE (lower figure) or biotinylated hK2 (1 μg / ml) and streptavidin-conjugated PE, CAR surface expression 24 hours after electroporation was measured by flow cytometry.

[0061] Figure 13 Shown is the cytotoxicity of hK2 CAR-T cells to hK2 positive (VCaP, top) and negative (DU145, bottom) tumor cells at a specified effector cell / target cell (E / T) ratio in a 20-hour flow-based assay. 24 hours after transient transfection, target cells were labeled with cell trace violet (CTV) fluorescent dye and then co-cultured with hK2 CAR-T cells. Simulated T cells served as negative effector controls. The percentage of killing was measured as the ratio of the absolute number of live (vitality dye negative) target (CTV positive) cells remaining in the co-culture to the number of live targets not cultured with CAR-T cells.

[0062] Figure 14 Real-time hK2 CAR-T cell-mediated cytotoxicity is shown. Normalized cell index (CI) charts of VCaP target cells (5E4) incubated for approximately 72 hours with simulation, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH or control CAR-T cells at different E: T ratios. When inoculated alone, target cells adhere to the plate and proliferate, increasing CI readings. When T cells are added to target cells, hK2 CAR and control CAR-T cells mediate hK2 positive VCaP cell lysis and CI subsequently decrease at E / T ratios of 5: 1 to 0.156: 1. The decrease in CI value after adding effector cells reflects the loss of viability of target cells. The Y-axis shows the normalized CI generated and displayed in real time by RTCA software. The X-axis is the cell culture time and treatment time in hours. The mean ± standard deviation of CI is plotted.

[0063] Figure 15Figure 24 shows the lack of cytotoxicity of target cells that do not express hK2 mediated by real-time hK2 CAR-T cells. Normalized cell index (CI) chart of DU145 target cells (5E3) incubated for approximately 72 hours with simulation, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH or control CAR-T cells at different E: T ratios. When inoculated alone, target cells adhere to the plate and proliferate, increasing CI readings. When T cells were added to target cells, hK2 CAR-T and control CAR-T cells did not reduce CI after addition and did not show cytolytic activity. The Y-axis shows the normalized CI generated and displayed in real time by RTCA software. The X-axis is the cell culture time and treatment time in hours. The mean ± standard deviation of CI is plotted.

[0064] Figure 16 Interferon-γ (IFN-γ) production of hK2 CAR-T cells stimulated by antigen is shown. Supernatants (VCap#5E4, DU145#5E3) were collected from the killing assay based on xCELLigence after about 70 hours of co-culture. hK2CAR_LH and control CAR modified T cells secreted IFN-γ during co-culture with hK2-expressing VCaP cells, but not during co-culture with hK2 negative DU145 cells. The average IFN-γ concentration ± standard deviation (pg / ml) from replicate cultures is shown.

[0065] Figure 17 Shown is the expression of hK2 CAR constructs (constructs 1-10) on Jurkat cells transduced with various hK2 CAR constructs as shown, containing a luciferase gene driven by a signaling-responsive NFAT promoter (JNL cells). Expression was determined using biotinylated hK2 followed by streptavidin-conjugated PE.

[0066] Figure 18 The combination between the CAR1-10 constructs and their homologous cell antigens (hK2 on target cells) detected by the luciferase expression in JNL cells after combination is shown. The JNL cells transduced with specified CAR constructs (CAR1-10) and the JNL cells (UTD) not transduced are co-cultured with target cell lines (VCaP or DU145 cells), and luciferase activity is measured according to luminous intensity. When luminous intensity exceeds 1.5 times of UTD cell levels in the presence of cells expressing antigens, it is believed that construct is active.

[0067] Figure 19Shown are hK2 CAR1-10 or parental 11B6_HL and 11B6_LH expression on the T cell surface. Primary human T cells were transduced with 11B6 thermostable and parental scFv CAR lentiviruses (multiplicity of infection (MOI) 3) and CAR expression was measured 14 days after transduction using biotinylated hK2 (1 μg / ml) followed by streptavidin-conjugated PE. UTD: untransduced control.

[0068] Figure 20 The percentage of tumor cell growth inhibition of hK2-positive VCaP cells by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH at an effector cell:target cell ratio of 1:1 or 0.5:1 is shown in a real-time IncuCyte killing assay assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of viable target cells - current number of viable target cells) / initial number of viable cells * 100 (%).

[0069] Figure 21 Shown are the percentages of tumor cell growth inhibition of PC3 cells by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH at an effector cell:target cell ratio of 1:1 in a real-time IncuCyte killing assay assessing antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of live target cells - current number of live target cells) / initial number of live cells * 100 (%).

[0070] Figure 22 Cytokine release of hK2 CAR-T cells is shown. The supernatant collected from the overnight (approximately 20 hours) co-culture of hK2 CAR-T cells and VCaP cells at an E / T ratio of 1: 1 was analyzed using a 13-plex Milliplex human high sensitivity T cell kit (HSTCMAG28SPMX13). hK2 CAR-T cells secrete cytokines during co-culture with hK2-expressing VCaP cells, but minimally secrete cytokines during co-culture with untransduced T cells (UTD). The average cytokine concentration ± standard deviation (pg / ml) from replicate cultures is shown.

[0071] Figure 23IFN-γ release of hK2 CAR-T cells is shown. Supernatant was collected from overnight (approximately 20 hours) co-cultures of hK2 CAR-T cells with VCap and DU145 cells (5E4 cells) at an E / T ratio of 1: 1. hK2 CAR-modified T cells secrete IFN-γ during co-culture with hK2-expressing VCaP cells, but not during co-culture with hK2-negative DU145 cells. The average IFN-γ concentration ± standard deviation (pg / ml) from replicate cultures is shown. T cells stimulated with CD3 / 28 beads and T cells only were used as positive and negative controls, respectively.

[0072] Figure 24A and Figure 24B Shown are flow cytometry histograms of Cell Trace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or CAR1-10-transduced CAR-T cells after 5 days of co-culture with VCaP or DU145 cells or after stimulation with CD3 / 28 beads.

[0073] Figure 25A and Figure 25B Shown are flow cytometry histograms of CD25-positive cell trace violet (CTV)-labeled untransduced T cells (T cells only in the figure) or CAR1-10-transduced CAR-T cells after 5 days of co-culture with VCaP or DU145 cells or after stimulation with CD3 / 28 beads.

[0074] Figure 26 hK2 CAR-T cells were shown to proliferate more robustly than CD3 / 28 bead-based positive controls after 5 days of co-culture with VCaP cells. T cells engineered with different CAR constructs exhibited varying proliferative activity and displayed varying CAR+ T cell counts. CAR+ T cell counts are based on mean absolute cell counts + / - SEM from three technical replicates.

[0075] Figure 27 Shown are the surface expressions of CAR17 (KL2B413_HL), CAR18 (KL2B413_LH), CAR19 (KL2B359_HL) and CAR20 (HK2B359_LH) on the surface of primary T cells. The numbers within each histogram indicate the percentage of cells expressing designated CARs.

[0076] Figure 28 Shown are the surface expression of CAR17 (KL2B413_HL), CAR18 (KL2B413_LH), CAR19 (KL2B359_HL), and CAR20 (KL2B359_LH) on JNL cells.

[0077] Figure 29The relative light units (RLU) generated by luciferase expression in JNL cells mediated by the binding of test CAR-T to its cognate receptor on various target cells as shown in the figure are shown. JNL cells containing the specified CAR clones and untransduced JNL cells (UTD) were co-cultured with target cell lines (VCaP, LNCaP / hK2, LNCaP, C4-2B, 22Rv1 or DU145 cells) and luciferase activity was measured according to luminescence intensity (RLU, relative light units). KL2B413_HL: CAR17, KL2B413_LH: CAR18, HL2B359_HL: CAR19, KL2B359_LH: CAR20.

[0078] Figure 30 The figure shows the percentage of tumor cell growth inhibition of hK2-positive VCaP cells by CAR-T cells transduced with CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time IncuCyte killing assay to assess antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of live target cells - current number of live target cells) / initial number of live cells * 100 (%).

[0079] Figure 31 The figure shows the percentage of tumor cell growth inhibition of hK2-negative DU145 cells by CAR-T cells transduced with CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time IncuCyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (initial number of live target cells - current number of live target cells) / initial number of live cells * 100 (%).

[0080] Figure 32 Shown are IFN-γ production by CAR-T cells transduced with CAR17 (KLK2B413HL in the figure), CAR18 (KLK2B413LH in the figure), CAR19 (KLK2B359HL in the figure), and CAR20 (KLK2B359LH in the figure), or untransduced T cells (UTD) when co-cultured with the indicated cells in the figure.

[0081] Figure 33A and Figure 33BIt was shown that co-culture of CAR-T cells transduced with CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure) and CAR20 (B359LH in the figure) and VCap cells resulted in an increase in CD107a+hK2-CAR-T+ cells, indicating immune cell activation and cytotoxic degranulation, whereas co-culture with hK2-negative DU145 cells had no effect.

[0082] Figure 34A and Figure 34B Flow cytometry histograms of untransduced T cells (T cells only in the figure) or CAR-T cells transduced with CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) labeled with Cell Trace Violet (CTV) after 5 days of co-culture with VCAp cells or DU145 cells. UTD: untransduced.

[0083] Figure 35 Shown are the percentages of proliferating cells in co-cultures of untransduced T cells (T cells only in the figure) or CAR-T cells transduced with CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) labeled with Cell Trace Violet (CTV) after 5 days of co-culture with VCAP cells or DU145 cells. UTD: untransduced.

[0084] Figure 36A and Figure 36B Shown are flow cytometry histograms of CTV+CD25+CAR-T cells transduced with CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCaP cells or DU145 cells for 5 days.

[0085] Figure 37 Shown are the percentages of untransduced T cells (UTD) expressing CTV or CAR-T cells transduced with CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after 5 days of co-culture with VCaP cells or DU145 cells, either alone (T cells only) or after stimulation with CD2 / 28 beads.

[0086] Figures 38A-38FThe binding paratopes of selected anti-hK2 antibodies and selected anti-hK2 / CD3 bispecific antibodies are shown. The underlined sequences indicate the CDR regions, and the highlighted sequences indicate the paratope regions. Figure 38A SEQ ID NOs 219 and 220 are disclosed in order of appearance, respectively. Figure 38B SEQ ID NOs 213 and 224 are disclosed in order of appearance, respectively. Figure 38C SEQ ID NOs 203 and 215 are disclosed in order of appearance, respectively. Figure 38D SEQ ID NOs 468 and 469 are disclosed in order of appearance, respectively. Figure 38E SEQ ID NOs 354 and 221 are disclosed in order of appearance, respectively. Figure 38F SEQ ID NOs 356 and 222 are disclosed in order of appearance, respectively.

[0087] Figure 39A Figure 2 shows the in vivo efficacy and cytokine profile of KLK2xCD3 bispecific antibodies in a VCaP xenograft mouse model. Three KLK2xCD3 bispecific antibodies were tested at three dose levels: 5 mg / kg, 1 mg / kg, and 0.2 mg / kg. Tumor growth inhibition was plotted based on tumor volume measurements.

[0088] Figure 39B Figure 2 shows the in vivo efficacy and cytokine profile of the KLK2xCD3 bispecific antibody in a VCaP xenograft mouse model. Six hours after the first dose, 100 μl blood samples were collected from the animals via retro-orbital bleeding. Plasma was separated from the blood samples by high-speed centrifugation. Luminex assays were performed to quantify IFN-γ and TNF-α concentrations at different KLK2 bispecific doses. DETAILED DESCRIPTION

[0089] The methods disclosed herein may be more readily understood with reference to the following detailed description in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the methods disclosed herein are not limited to the specific methods described and / or illustrated herein, and that the terminology used herein is intended only to describe specific embodiments by way of example and is not intended to limit the methods protected by the claims.

[0090] All patents, published patent applications, and publications cited herein are incorporated by reference as if fully set forth herein.

[0091] When a list is provided, it is understood that each individual element of the list and each combination of the list is a separate embodiment, unless otherwise indicated. For example, a list of embodiments presented as "A, B, or C" will be understood to include embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0092] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes reference to two or more cells, and so forth.

[0093] The transition terms "comprising," "consisting essentially of," and "consisting of are intended to suggest their recognized meanings in patent parlance; that is, (i) "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. Also provided are embodiments described with the phrase "comprising" (or its equivalents), as do those described independently with "consisting of" and "consisting essentially of."

[0094] "About" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless expressly indicated otherwise in the examples or elsewhere in the specification, "about" means within one standard deviation, or up to 5%, whichever is greater, according to customary practice in the art.

[0095] "Activation" or "stimulation" or "activated" or "stimulated" refers to the induction of a change in the biological state of a cell, resulting in expression of activation markers, cytokine production, target cell proliferation, or mediation of target cell cytotoxicity. A cell can be activated by a primary stimulatory signal. A co-stimulatory signal can amplify the amplitude of the primary signal and suppress cell death after the initial stimulation, thereby producing a more persistent activation state and therefore a higher cytotoxic capacity. A "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR / CD3 ligation), results in T cell and / or NK cell proliferation and / or upregulation or downregulation of key molecules.

[0096] "Alternative scaffolds" refer to single-chain protein frameworks comprising a structured core associated with highly conformationally tolerant variable domains. The variable domains tolerate changes to be introduced without compromising scaffold integrity, and thus can be engineered and selected to bind to specific antigens.

[0097] "Antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refers to a mechanism of cell death induction that relies on the interaction of antibody-coated target cells with lytic effector cells (such as natural killer (NK) cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγRs) expressed on the effector cells.

[0098] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes, such as macrophages or dendritic cells.

[0099] "Antigen" refers to any molecule (e.g., a protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portion thereof, or combination thereof) that can be bound by an antigen binding domain or a T cell receptor that can mediate an immune response. Exemplary immune responses include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens can be genetically expressed, synthesized, or purified from a biological sample (such as a tissue sample, a tumor sample, a cell or fluid with other biological components, an organism, a subunit of a protein / antigen, killed or inactivated whole cells, or a lysate).

[0100] "Antigen-binding fragment" or "antigen-binding domain" refers to a portion of a protein that binds an antigen. Antigen-binding fragments can be synthetic, enzymatically obtainable, or genetically engineered polypeptides and include antigen-binding portions of immunoglobulins, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd, and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain, shark variable Ig NAR domains, humped VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDRs of an antibody, such as the FR3-CDR3-FR4 portion, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind antigen, and multispecific proteins comprising antigen-binding fragments. Antigen binding fragments (such as VH and VL) can be linked together via synthetic linkers to form various types of single antibody designs, wherein in those cases where the VH and VL domains are expressed by separate single chains, the VH / VL domains can be paired intramolecularly or intermolecularly to form a monovalent antigen binding site, such as single-chain Fv (scFv) or diabodies. Antigen binding fragments can also be conjugated to other antibodies, proteins, antigen binding fragments, or alternative scaffolds, which can be monospecific or multispecific to engineer bispecific and multispecific proteins.

[0101] "Antibody" refers broadly to and includes immunoglobulin molecules, specifically monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies and chimeric monoclonal antibodies), antigen-binding fragments, multispecific antibodies such as bispecific, trispecific or tetraspecific antibodies, dimeric, tetrameric or multimeric antibodies, single-chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecules that contain an antigen-binding site with the desired specificity. "Full-length antibodies" comprise two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (composed of the domains CH1, hinge, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions, which are called complementarity determining regions (CDRs) and are interspersed with framework regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulins can be assigned to five major classes based on the amino acid sequence of the heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subclassified into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct classes, kappa and lambda, based on the amino acid sequence of their constant domains.

[0102] "Bispecific" refers to a molecule (such as an antibody) that specifically binds to two different antigens or two different epitopes within the same antigen. Bispecific molecules may have cross-reactivity to other related antigens, for example, to the same antigen from another species (homologs), such as humans or monkeys, e.g., cynomolgus (cyno) or chimpanzees (Pan troglodytes), or may bind to an epitope shared between two or more different antigens.

[0103] "Bispecific anti-hK2 / anti-CD3 antibody", "hK2 / CD3 antibody", "hk2xCD3 antibody", "anti-hK2 / anti-CD3 protein", etc. refers to an antibody that binds hK2 and CD3 and comprises at least one binding domain that specifically binds hK2 and at least one binding domain that specifically binds CD3. The domains that specifically bind hK2 and CD3 are typically V H / V L Yes. The bispecific anti-HK2×CD3 antibody can be monovalent with respect to its binding to HK2 or CD3.

[0104] "Cancer" refers to a broad variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors.

[0105] As used herein, a "chimeric antigen receptor" (CAR) is defined as a cell surface receptor comprising an extracellular target binding domain, a transmembrane domain, and an intracellular signaling domain, all of which are not naturally present on a single protein. This includes receptors in which the extracellular domain and the intracellular signaling domain do not naturally occur together on a single receptor protein. CARs are primarily intended for use in lymphocytes, such as T cells and natural killer (NK) cells.

[0106] "Complement-dependent cytotoxicity" or "CDC" refers to a mechanism of cell death induction in which the Fc effector domain of a target-binding protein binds and activates the complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote CDC by binding to complement receptors (e.g., CR3) on leukocytes.

[0107] "Complementarity determining regions" (CDRs) are regions of antibodies that bind antigen. There are three CDRs in VH (HCDR1, HCDR2, HCDR3) and three CDRs in VL (LCDR1, LCDR2, LCDR3). CDRs can be defined using various delineations, such as Kabat (Wu et al., (1970) J Exp Med 132:211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al., (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton, J Bmol Biol 263:800-15, 1996). The correspondence between various delineations and variable region numbering is described (see, e.g., Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-70; International Immunogenetics (IMGT) database; Web resource, http: / / www_imgt_org). Available programs (such as abYsis from UCL Business PLC) can be used to delineate CDRs. Unless otherwise expressly stated in the specification, as used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include CDRs defined by any of the above methods (Kaba, Chothia, IMGT or AbM).

[0108] "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR) complex and composed of homodimers or heterodimers formed by the association of two or four receptor chains: CD3ε, CD3δ, CD3ζ, and CD3γ. Human CD3ε comprises the amino acid sequence of SEQ ID NO: 442. Unless expressly indicated as being from a non-human species, all references herein to proteins, polypeptides, and protein fragments are intended to refer to the human form of the corresponding protein, polypeptide, or protein fragment. Thus, unless indicated as being from a non-human species, e.g., "mouse CD3" or "monkey CD3," etc., "CD3" refers to human CD3.

[0109] Throughout this specification, "CD3-specific," "specifically binds to CD3," or "anti-CD3 antibody" refers to antibodies that specifically bind to the CD3-ε polypeptide (SEQ ID NO: 442), including antibodies that specifically bind to the CD3-ε extracellular domain (ECD) (SEQ ID NO: 443). CD3-ε, along with CD3-γ, -δ, and -ζ, and T cell receptor α / β and γ / δ heterodimers, forms the T cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signaling pathways. The CD3 complex mediates signaling leading to T cell activation and proliferation. CD3 is required for immune responses.

[0110] SEQ ID NO: 442 (human CD3ε)

[0111]

[0112] SEQ ID NO: 443 (human CD3ε extracellular domain)

[0113]

[0114] "Decrease", "lower", "mitigate", "reduce" or "decrease" generally refers to the ability of a test molecule to mediate a reduced response (i.e., a downstream effect) when compared to a response mediated by a control or vehicle. Exemplary responses are T cell expansion, T cell activation or T cell-mediated killing of tumor cells or binding of a protein to its antigen or receptor, enhanced binding to Fcγ or enhanced Fc effector function, such as enhanced ADCC, CDC and / or ADCP. A decrease can be a statistically significant difference in the response measured between the test molecule and a control (or vehicle), or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, such as 500, 600, 700, 800, 900, or 1000-fold or more (including all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0115] "Differentiation" refers to a process of reducing the potential or proliferation of a cell or moving a cell to a more developmentally restricted state.

[0116] The term "encode" or "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, if transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene, cDNA, or RNA encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence) and the non-coding strand (which serves as a template for transcription of the gene or cDNA) can be said to encode the protein, or other product of the gene or cDNA.

[0117] "Enhance," "promote," "increase," "amplify," or "improve" generally refers to the ability of a test molecule to mediate a stronger response (i.e., a downstream effect) when compared to a response mediated by the test molecule or vehicle. Exemplary responses are T cell expansion, T cell activation, or T cell-mediated killing of tumor cells or binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector function, such as enhanced ADCC, CDC, and / or ADCP. Enhancement can be a statistically significant difference in the response measured between the test molecule and a control (or vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, such as 500, 600, 700, 800, 900, or 1000-fold or more (including all integers and decimal points between and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0118] "Epitope" refers to the portion of an antigen to which an antibody specifically binds. An epitope is typically composed of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups of moieties such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be composed of continuous and / or discontinuous amino acids that form a unit of conformational space. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are close in three-dimensional space due to the folding of the protein molecule. The antibody "epitope" depends on the method used to identify the epitope.

[0119] "Amplification" refers to the result of cell division and cell death.

[0120] "Express" and "expression" refer to the well-known transcription and translation that occur in or outside a cell. An expression product, such as a protein, is expressed by the cell or expressed in vitro and can be an intracellular protein, an extracellular protein, or a transmembrane protein.

[0121] "Expression vector" refers to a vector that can be used in a biological system or a reconstructed biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0122] "dAb" or "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341: 544-546 (1989)).

[0123] "Fab" or "Fab fragment" refers to an antibody fragment consisting of the VH, CH1, VL, and CL domains.

[0124] "F(ab')2" or "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments linked by a disulfide bridge in the hinge region.

[0125] "Fd" or "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains.

[0126] "Fv" or "Fv fragment" refers to an antibody fragment consisting of the VH and VL domains from a single arm of an antibody. An Fv fragment lacks the constant regions of the Fab (CH1 and CL) region. The VH and VL in the Fv fragment are held together by non-covalent interactions.

[0127] The "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain. For example, the Fc polypeptide of a dimeric IgG Fc comprises the IgG CH2 and IgG CH3 constant domain sequences).

[0128] "Full-length antibodies" are composed of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds and their multimers (e.g., IgM). Each heavy chain is composed of a heavy chain variable domain (VH) and a heavy chain constant domain, and the heavy chain constant domain is composed of the subdomains CH1, hinge, CH2, and CH3. Each light chain is composed of a light chain variable domain (VL) and a light chain constant domain (CL). VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL is composed of three CDRs and four FR segments, and is arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0129] "Genetic modification" refers to the introduction of a "foreign" (i.e., external or extracellular) gene, DNA or RNA sequence into a host cell so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be referred to as a "cloned" or "foreign" gene or sequence, and may include regulatory or control sequences operably linked to a polynucleotide encoding a chimeric antigen receptor, such as a start sequence, stop sequence, promoter sequence, signal sequence, secretion sequence or other sequence used by the cell's genetic machinery. The gene or sequence may include non-functional sequences or sequences with no known function. A host cell that receives and expresses the introduced DNA or RNA has been "genetically engineered." The DNA or RNA introduced into a host cell may come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0130] "Heterologous" refers to two or more polynucleotides or two or more polypeptides that are not found in the same relationship to each other in nature.

[0131] "Heterologous polynucleotide" refers to a non-naturally occurring polynucleotide that encodes two or more neoantigens as described herein.

[0132] "Heterologous polypeptide" refers to a non-naturally occurring polypeptide comprising two or more neoantigenic polypeptides as described herein.

[0133] "Host cell" refers to any cell that contains heterologous nucleic acid. An exemplary heterologous nucleic acid is a vector (eg, an expression vector).

[0134] "Human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable regions of human antibodies are derived from human immunoglobulin sequences. If a human antibody comprises a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. If the variable regions of a human antibody are obtained by a system using human germline immunoglobulins or rearranged immunoglobulin genes, the human antibody comprises a heavy chain variable region and a light chain variable region that are "derived from" sequences of human origin. Exemplary systems of this type are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals, such as mice or rats carrying human immunoglobulin loci. Due to the differences between the systems used to obtain human antibodies and human immunoglobulin loci, the introduction of somatic mutations or the deliberate introduction of substitutions into the framework or CDRs or both, "human antibodies" typically contain amino acid differences compared to immunoglobulins expressed in humans. Ordinarily, a "human antibody" has an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence encoded by a human germline immunoglobulin gene or a rearranged immunoglobulin gene. In some cases, a "human antibody" may comprise a consensus framework sequence derived from human framework sequence analysis (e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86), or a synthetic HCDR3 bound to a human immunoglobulin gene library displayed on phage (e.g., as described in Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO2009 / 085462). The definition of "human antibody" does not include antibodies in which at least one CDR is derived from a non-human species.

[0135] "Humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Humanized antibodies may contain substitutions in the framework so that the framework may not be an exact copy of an expressed human immunoglobulin or a human immunoglobulin germline gene sequence.

[0136] "In combination with" means that two or more therapeutic agents are administered to a subject together in a mixture, simultaneously as a single agent, or sequentially in any order as a single agent.

[0137] "Intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of a molecule. The functional portion of the protein acts by transmitting information within the cell to regulate cellular activity via a defined signaling pathway by generating second messengers, or to act as an effector by responding to such messengers. The intracellular signaling domain generates signals that promote the immune effector functions of CAR-containing cells (e.g., CAR-T cells).

[0138] "Isolated" refers to a homogenous population of molecules (such as synthetic polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of a system (such as a recombinant cell) in which the molecule is produced, as well as proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals, and encompasses molecules isolated to higher degrees of purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0139] "Kallikrein-related peptidase 2" or "hK2" refers to a known protein also known as kallikrein-2, adenylyl peptidase 2, or HK2. hK2 is produced as a preproprotein and is cleaved during proteolysis to generate the active protease. All hK2 isoforms and variants are encompassed by "hK2." The amino acid sequences of the various isoforms can be retrieved from GenBank Accession Nos. NP_005542.1, NP_001002231.1, and NP_001243009. The amino acid sequence of full-length hK2 is shown in SEQ ID NO: 62. This sequence includes a signal peptide (residues 1-18) and a propeptide region (residues 19-24).

[0140] SEQ ID NO: 62

[0141]

[0142]

[0143] "Modulate" refers to the ability of a test molecule to mediate an increase or decrease in a response (ie, a downstream effect) when compared to the response mediated by a control or vehicle.

[0144] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of the C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation. Monoclonal antibodies generally bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, monovalent, bivalent, or multivalent.

[0145] "Multispecific" refers to a molecule, such as an antibody, that specifically binds to two or more different antigens or two or more different epitopes within the same antigen. A multispecific molecule may have cross-reactivity to other related antigens, for example, to the same antigen from another species (homologous), such as human or monkey, e.g., cynomolgus (cyno) or chimpanzee, or may bind to an epitope shared between two or more different antigens.

[0146] "Natural killer cells" and "NK cells" are used interchangeably and synonymously herein. NK cells are cells with CD16 + CD56 + and / or CD57 + Differentiated lymphocytes with a TCR-phenotype. NK cells are characterized by their ability to bind to and kill cells that do not express "self" MHC / HLA antigens by activating specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or suppress immune responses.

[0147] "Operably linked" and similar phrases, when used with reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequences, respectively, that are placed in a functional relationship with each other. For example, an operably linked promoter, enhancer element, open reading frame, 5' and 3' UTRs, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA), and in some cases, a polypeptide (i.e., expression of the open reading frame). An operably linked peptide is one in which the functional domains of the peptide are placed at an appropriate distance from each other to confer the intended function of each domain.

[0148] The term "paratope" refers to an area or region of an antibody molecule that is involved in antigen binding and comprises residues that interact with the antigen. The paratope can be composed of continuous and / or discontinuous amino acids that form a unit of conformational space. Various experimental and computational methods can be used to define and characterize the paratope of a given antibody with different levels of detail. Experimental methods include hydrogen / deuterium exchange mass spectrometry (HX-MS). Depending on the mapping method used, the paratope will be defined differently.

[0149] A "pharmaceutical combination" refers to a combination of two or more active ingredients, administered together or separately.

[0150] "Pharmaceutical composition" refers to a composition resulting from the combination of active ingredients and a pharmaceutically acceptable carrier.

[0151] "Pharmaceutically acceptable carrier" or "excipient" refers to an ingredient in a pharmaceutical composition other than the active ingredient, which is non-toxic to the subject. Exemplary pharmaceutically acceptable carriers are buffers, stabilizers, or preservatives.

[0152] "Polynucleotide" or "nucleic acid" refers to a synthetic molecule comprising a chain of nucleotides covalently linked to a phosphate sugar backbone or other equivalent covalent chemical bonds. cDNA is a typical example of a polynucleotide. A polynucleotide can be a DNA or RNA molecule.

[0153] "Prevent," "preventing," "prevention," or "prophylaxis" of a disease or condition means preventing the condition from occurring in a subject.

[0154] "Proliferation" refers to an increase in cell division, either symmetrical or asymmetrical.

[0155] "Promoter" refers to the minimal sequence required to initiate transcription. A promoter may also include enhancer or repressor elements that increase or repress transcription, respectively.

[0156] "Protein" or "polypeptide" are used interchangeably herein to refer to a molecule comprising one or more polypeptides, each comprising at least two amino acid residues linked by peptide bonds. A protein can be a monomer or a protein complex of two or more subunits, which can be identical or different. Small polypeptides of less than 50 amino acids can be referred to as "peptides." A protein can be a heterologous fusion protein, a glycoprotein, or a protein modified by a post-translational modification, such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation, or biotinylation. A protein can be recombinantly expressed.

[0157] "Recombinant" refers to polynucleotides, polypeptides, vectors, viruses, and other macromolecules that are prepared, expressed, created, or isolated by recombinant means.

[0158] "Regulatory element" refers to any cis- or trans-acting genetic element that controls some aspect of the expression of a nucleic acid sequence.

[0159] "Relapse" refers to the reappearance of a disease or signs and symptoms of a disease after a period of improvement following previous treatment with a therapeutic agent.

[0160] "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment before or at the start of treatment, or a refractory disease may become resistant during treatment.

[0161] "Single-chain Fv" or "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region (VL) and at least one antibody fragment containing a heavy chain variable region (VH), wherein the VL and the VH are continuously connected via a polypeptide linker and are capable of being expressed as a single-chain polypeptide. Unless otherwise indicated, as used herein, scFv can have the VL variable region and the VH variable region in either order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.

[0162] "(scFv)2" or "tandem scFv" or "double scFv" fragment" refers to a fusion protein comprising two light chain variable regions (VL) and two heavy chain variable regions (VH), wherein the two VL and two VH regions are continuously connected via a polypeptide linker and can be expressed as a single polypeptide chain. The two VL and two VH regions fused by a peptide linker form a bivalent molecule VL A -Connector-VH A -Connector-VL B -Connector-VH B To form two binding sites that can simultaneously bind to two different antigens or epitopes.

[0163] "Specifically binds", "specific binding", "specifically binding" or "binds" refers to the binding of a proteinaceous molecule to an antigen or an epitope within an antigen with a greater affinity than to other antigens. Typically, a proteinaceous molecule binds to an antigen or an epitope within an antigen with an affinity of about 1×10 -7 M or smaller (e.g., about 5×10 -8 M or smaller, approximately 1×10 -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10M or smaller, approximately 1×10 -11 M or smaller or about 1×10 -12 or smaller) of the equilibrium dissociation constant (K D ) binds to an antigen or an epitope within an antigen, typically with a KD at least one hundred-fold less than its KD for binding to a nonspecific antigen (e.g., BSA, casein). In the context of the prostate neoantigens described herein, "specific binding" refers to binding of a proteinaceous molecule to the prostate neoantigen without detectable binding to the wild-type protein of which the neoantigen is a variant.

[0164] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms "subject" and "patient" are used interchangeably herein.

[0165] "T cells" and "T lymphocytes" are interchangeable and used synonymously herein. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 + T cells), CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + Cells or any other subpopulation of T cells. Also included are "NKT cells", which refer to a population of specialized T cells that express semi-invariant αβ T cell receptors but also express various molecular markers commonly associated with NK cells (such as NK1.1). NKT cells include NK1.1 + and NK1.1 - and CD4 + 、CD4 - 、CD8 + and CD8 -Cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by MHC I-like molecules CD Id. Since NKT cells can produce cytokines that promote inflammation or immune tolerance, they can have protective or harmful effects. Also included are "γδ T cells", which refer to a small number of specialized populations of T cells with different TCRs on their surface, and unlike most T cells in which the TCR is composed of two glycoprotein chains named α- and β-TCR chains, the TCR in γδ T cells is composed of a γ chain and a δ chain. γδ T cells can play a role in immune surveillance and immune regulation, and have been found to be an important source of IL-17 and to induce powerful CD8 + Cytotoxic T cell responses. Also included are "regulatory T cells" or "Tregs," which are T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance. Tregs are typically CD4+ cells that are positive for the transcription factor Foxp3. + T cells, and may also include transcription factor Foxp3-negative regulatory T cells, which are CD4 + T cells.

[0166] "Therapeutically effective amount" or "effective amount" are used interchangeably herein and refer to an amount effective to achieve the desired therapeutic outcome, at dosages and for periods of time necessary. A therapeutically effective amount may vary according to factors such as the individual's disease state, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improvement in the patient's health status, reduction in tumor burden, arrest or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body.

[0167] "Transduction" refers to the introduction of foreign nucleic acid into cells using viral vectors.

[0168] "Treat," "treating," or "treatment" of a disease or disorder, such as cancer, means achieving one or more of the following: reducing the severity and / or reducing the duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who previously had symptoms of the disorder.

[0169] "Tumor cell" or "cancer cell" refers to a cancerous, precancerous or transformed cell in vivo, ex vivo or in tissue culture that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. However, transformation can occur by infection with a transforming virus and the incorporation of new genomic nucleic acid, the uptake of exogenous nucleic acid, or it can occur spontaneously or after exposure to a carcinogen, thereby mutating endogenous genes. Transformation / cancer is exemplified by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignancy, modulation of tumor-specific marker levels, invasion, tumor growth in a suitable animal host (such as nude mice, etc.) in vitro, in vivo and ex vivo.

[0170] "Variant," "mutant," or "altered" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, such as one or more substitutions, insertions, or deletions.

[0171] Unless otherwise specifically indicated, throughout the specification, amino acid residues in the antibody constant region are numbered according to 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).

[0172] Mutations in the Ig constant region are designated as follows: L351Y_F405A_Y407V, which refers to the L351Y, F405A, and Y407V mutations in one immunoglobulin constant region. L351Y_F405A_Y407V / T394W refers to the L351Y, F405A, and Y407V mutations in the first Ig constant region and the T394W mutation in the second Ig constant region present in a multimeric protein.

[0173] "VHH" refers to a single domain antibody or nanobody composed only of the antigen binding domain of the heavy chain. VHH single domain antibodies lack the light chain and the CH1 domain of the heavy chain of the conventional Fab region.

[0174] Composition of matter

[0175] hK2-binding antigen-binding domain

[0176] The present disclosure provides antigen binding domains that bind to hK2, monospecific and multispecific proteins (particularly bispecific proteins) comprising antigen binding domains that bind to hK2, chimeric antigen receptors (CARs) comprising antigen binding domains that bind to hK2, polynucleotides encoding the foregoing, vectors, host cells, and methods of preparing and using the foregoing. The antigen binding domains that bind to hK2 identified herein exhibit improved properties in terms of improved thermal stability. The multispecific proteins disclosed herein may be particularly effective in mediating T cell-mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release, and / or exhibiting increased anti-tumor efficacy.

[0177] The present disclosure provides an isolated protein comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the hK2-binding antigen binding domain binds to an epitope on hK2 set forth in SEQ ID NO: 111, 112, 113, 114, or 115.

[0178] In a specific embodiment, the hK2 binding antigen binding domain binds to the epitope set forth in SEQ ID NOs: 111 and 112 on hK2.

[0179] In some embodiments, the hK2 binding antigen binding domain binds to the epitope set forth in SEQ ID NOs: 113, 114, and 115 on hK2.

[0180] The present invention also provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antibody or antigen binding fragment thereof binds within residues KVTEF (SEQ ID NO: 111) or HYRKW (SEQ ID NO: 112) or SHGWAH (SEQ ID NO: 113) or RHNLFEPEDTGQRVP (SEQ ID NO: 114) or GWGSIEPEE (SEQ ID NO: 115) of hK2. In a specific embodiment, the present invention also provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain binds to hK2 within an epitope having the sequences of KVTEF (SEQ ID NO: 111) and HYRKW (SEQ ID NO: 112).

[0181] The residues within hK2 that bind to the antibody can be determined using an H / D exchange assay. In the H / D exchange assay, recombinantly expressed soluble hK2 is incubated in deuterated water in the presence or absence of an antibody for a predetermined time, resulting in the introduction of deuterium at exchangeable hydrogen atoms that are not protected by the antibody, after which the protein is digested with a protease and the peptide fragments are analyzed using LC-MS. The H / D exchange assay can be performed using known protocols. An exemplary protocol is described in Example 3. In some embodiments, the H / D exchange mixture is quenched by adding a quenching buffer (e.g., 8 M urea, 1 M TCEP, pH 3.0) before being passed through an equilibrated immobilized pepsin / FPXIII column at room temperature (e.g., 600 μL / min). The peptide fragments are then loaded onto a reverse phase trap column (e.g., 600 μL / min) and desalted (e.g., at 600 μL for 1 min), separated (e.g., on a C18 column), and analyzed by mass spectrometry (e.g., using an LTQ TM Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) with a capillary temperature of 275 °C, a resolution of 150,000 and a mass range (m / z) 300–1,800).

[0182] The present invention also provides an isolated protein comprising an antigen binding domain, wherein the antigen binding domain of the reference antibody binds to an epitope on hK2 having a sequence selected from the group consisting of SEQ ID NOs: 111, 112, 113, 114, and 115, and wherein the antigen binding domain that binds hK2 competes with the reference antibody disclosed herein for binding to hK2. In some embodiments, the reference antibody comprises:

[0183] a. HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 137 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 138; or

[0184] b. HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 162 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 163; or

[0185] c. HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 164 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 165; or

[0186] d. HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 166 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 167; or

[0187] e. HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 168 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 169; or

[0188] f. HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 204 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 205; or

[0189] g. HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 166 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 444.

[0190] In certain such embodiments, the reference antibody comprises heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 162 and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 163.

[0191] Competition for binding of a test antibody that binds to soluble hK2 of SEQ ID NO: 111, 112, 113, 114, or 115 with a reference antibody of the invention can be determined in vitro using well-known methods. For example, binding of a labeled hK2 antibody to the hK2 membrane-proximal region in the presence of an unlabeled reference antibody can be assessed by ELISA, or competition can be demonstrated using Bioacore analysis or flow cytometry. A test antibody competes with the reference antibody for hK2 binding when the test antibody inhibits binding of the reference antibody to soluble hK2 by 85% or greater, e.g., 90% or greater, or 95% or greater.

[0192] The present disclosure provides an isolated protein comprising an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises:

[0193] or HCDR1, HCDR2, and HCDR3 of a VH of SEQ ID NO: 162 and LCDR1, LCDR2, and LCDR3 of a VL of SEQ ID NO: 163; or HCDR1, HCDR2, and HCDR3 of a VH of SEQ ID NO: 164 and LCDR1, LCDR2, and LCDR3 of a VL of SEQ ID NO: 165; or HCDR1, HCDR2, and HCDR3 of a VH of SEQ ID NO: 166 and LCDR1, LCDR2, and LCDR3 of a VL of SEQ ID NO: 167. or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 168 and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 169; or HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 204 and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 205. In a specific embodiment, the isolated protein comprises an antigen binding domain that binds kallikrein-related peptidase 2 (hK2), wherein the antigen binding domain that binds hK2 comprises HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 162 and LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 163.

[0194] The present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[0195] SEQ ID NOs: 63, 65, 66, 67, 69, 71, respectively;

[0196] SEQ ID NOs: 63, 65, 66, 68, 70, 71, respectively;

[0197] SEQ ID NOs: 72, 73, 66, 67, 69, 71, respectively;

[0198] SEQ ID NOs: 141, 142, 143, 144, 145 and 146, respectively;

[0199] SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively;

[0200] They are SEQ ID NOs: 176, 177, 178, 179, 180 and 181, respectively.

[0201] SEQ ID NOs: 170, 183, 184, 185, 186 and 187, respectively;

[0202] SEQ ID NOs: 188, 189, 190, 191, 192 and 193, respectively;

[0203] SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively;

[0204] SEQ ID NOs: 147, 148, 143, 144, 145 and 146, respectively;

[0205] SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively;

[0206] SEQ ID NOs: 196, 197, 178, 179, 180, 181, respectively;

[0207] SEQ ID NOs: 198, 199, 184, 185, 186 and 187, respectively;

[0208] SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively; or

[0209] They are SEQ ID NOs: 216, 217, 218, 182, 470 and 209, respectively.

[0210] In a specific embodiment, the present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively.

[0211] In another specific embodiment, the present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively.

[0212] The present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 137, 162, 164, 166, 168 or 204 and the VL of SEQ ID NO: 138, 163, 165, 167 or 169.

[0213] The present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises:

[0214] VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138;

[0215] VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163;

[0216] VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165;

[0217] VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167;

[0218] VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169; or

[0219] The VH of SEQ ID NO: 204 and the VL of SEQ ID NO: 205.

[0220] In a specific embodiment, the disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163.

[0221] The present disclosure also provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0222] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0223] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0224] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 95% identical to the VH of SEQ ID NO:162 and a VL that is at least 95% identical to the VL of SEQ ID NO:163.

[0225] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 95% identical to the VH of SEQ ID NO:162 and a VL that is at least 99% identical to the VL of SEQ ID NO:163.

[0226] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 99% identical to the VH of SEQ ID NO:162 and a VL that is at least 99% identical to the VL of SEQ ID NO:163.

[0227] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 99% identical to the VH of SEQ ID NO:162 and a VL that is at least 95% identical to the VL of SEQ ID NO:163.

[0228] The present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO: 133, 134, 308, 316, 324, 325, 404, 405, 406, 407, 408, or 409. In a specific embodiment, the present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO: 404.

[0229] In another embodiment, the present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:405.

[0230] The present disclosure also provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 404.

[0231] The present disclosure also provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 405.

[0232] The present disclosure also provides an isolated protein comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), wherein the antigen-binding domain that binds to hK2 comprises a heavy chain variable region (VH) of SEQ ID NO: 75 and a light chain variable region (VL) of SEQ ID NO: 74. SEQ ID NO: 75 and SEQ ID NO: 74 represent genera encompassing VH and VL amino acid sequences of variants exhibiting improved thermal stability compared to the parent antibody hu11B6. The engineered positions that confer improved thermal stability are residues P41, I49, M70, and A88 in the VH (residue numbering is according to hu11B6_VH of SEQ ID NO: 5) and residues S80, L82, A88, and Y91 in the VL (residue numbering is according to hu11B6_VL of SEQ ID NO: 2).

[0233] SEQ ID NO: 75 (VH consensus sequence)

[0234]

[0235] SEQ ID NO: 74 (VL consensus sequence)

[0236]

[0237] The present disclosure provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 4, 5, 6, 139, 159 or 161 and a VL of SEQ ID NO: 1, 2, 3, 140 or 160, with the proviso that the antigen binding domain that binds hK2 does not comprise both a VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 2.

[0238] The present disclosure also provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises:

[0239] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1;

[0240] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2;

[0241] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3;

[0242] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140;

[0243] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160;

[0244] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1;

[0245] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3;

[0246] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140;

[0247] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160;

[0248] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1;

[0249] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2;

[0250] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3;

[0251] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140;

[0252] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160;

[0253] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1;

[0254] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2;

[0255] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3;

[0256] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140;

[0257] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 160;

[0258] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1;

[0259] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2;

[0260] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3;

[0261] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140;

[0262] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160;

[0263] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1;

[0264] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2;

[0265] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3;

[0266] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or

[0267] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 160.

[0268] The present disclosure also provides an isolated protein comprising an antigen binding domain that binds hK2, wherein the antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 135, 136, 318, 319, 320, 321, 322, or 323.

[0269] In some embodiments, the antigen binding domain that binds hK2 is a scFv.

[0270] In some embodiments, the antigen binding domain that binds hK2 is (scFv)2.

[0271] In some embodiments, the antigen binding domain that binds hK2 is an Fv.

[0272] In some embodiments, the antigen binding domain that binds hK2 is a Fab.

[0273] In some embodiments, the antigen binding domain that binds hK2 is F(ab')2.

[0274] In some embodiments, the antigen binding domain that binds hK2 is Fd.

[0275] In some embodiments, the hK2 antigen binding domain is a dAb.

[0276] In some embodiments, the hK2 antigen binding domain is a VHH.

[0277] In a specific embodiment, the antigen binding domain that binds hK2 is a Fab.

[0278] hK2-binding scFv

[0279] Any of the hK2-binding VH and VL domains identified herein can be engineered into scFv formats in either the VH-linker-VL or VL-linker-VH orientations. Any of the VH and VL domains identified herein can also be used to generate sc(Fv)2 structures such as VH-linker-VL-linker-VL-linker-VH, VH-linker-VL-linker-VH-linker-VL, VH-linker-VH-linker-VL-linker-VL, VL-linker-VH-linker-VH-linker-VL, VL-linker-VH-linker-VH-linker-VL, VL-linker-VH-linker-VH, VL-linker-VL-linker-VH-linker-VH, or VL-linker-VL-linker-VH-linker-VH.

[0280] The VH and VL domains identified herein can be incorporated into scFv formats, and the resulting scFvs can be evaluated for binding and thermal stability to hK2 using known methods. Binding can be assessed using a ProteOn XPR36, BIAcore3000, or KinExA instrument, ELISA, or competitive binding assays known to those skilled in the art. Binding can be evaluated using purified scFv or E. coli supernatant or lysed cells containing expressed scFv. The affinity of the measured test scFv for hK2 may vary if measured under different conditions (e.g., osmolarity, pH). Therefore, affinity and other binding parameters (e.g., K) can be evaluated. D , K on , K off ) are typically measured using standardized conditions and standardized buffers. Thermal stability can be assessed by heating a test scFv at an elevated temperature, such as 50°C, 55°C, or 60°C, for a period of time, such as 5 minutes (min), 10 min, 15 min, 20 min, 25 min, or 30 min, and measuring the binding of the test scFv to hK2. ScFvs that maintain equivalent binding to hK2 when compared to unheated scFv samples are considered thermally stable.

[0281] In recombinant expression systems, the linker is a peptide linker and can comprise any naturally occurring amino acid. Exemplary amino acids that can be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The length of the linker should be sufficient to connect the VH and VL in the correct conformation relative to each other so that they retain the desired activity (such as binding to hK2).

[0282] The linker may be about 5 to 50 amino acids long. In some embodiments, the linker is about 10 to 40 amino acids long. In some embodiments, the linker is about 10 to 35 amino acids long. In some embodiments, the linker is about 10 to 30 amino acids long. In some embodiments, the linker is about 10 to 25 amino acids long. In some embodiments, the linker is about 10 to 20 amino acids long. In some embodiments, the linker is about 15 to 20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids in length. In some embodiments, the linker is 18 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker is 22 amino acids in length. In some embodiments, the linker is 23 amino acids in length. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 26 amino acids in length. In some embodiments, the linker is 27 amino acids in length. In some embodiments, the linker is 28 amino acids in length. In some embodiments, the linker is 29 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 31 amino acids in length. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker is 33 amino acids in length. In some embodiments, the linker is 34 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is 36 amino acids in length. In some embodiments, the length of the linker is 37 amino acids. In some embodiments, the length of the linker is 38 amino acids. In some embodiments, the length of the linker is 39 amino acids. In some embodiments, the length of the linker is 40 amino acids.Exemplary linkers that can be used are Gly-rich linkers, linkers containing Gly and Ser, linkers containing Gly and Ala, linkers containing Ala and Ser, and other flexible linkers.

[0283] Other linker sequences can include portions of immunoglobulin hinge regions, CL or CH1 derived from any immunoglobulin heavy or light chain isotype. Alternatively, a variety of non-protein polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol can be used as linkers. Exemplary linkers that can be used are shown in Table 1. Additional linkers are described, for example, in International Patent Publication No. WO2019 / 060695.

[0284] In some embodiments, the scFv comprises, from N-terminus to C-terminus, a VH, a first linker (L1), and a VL (VH-L1-VL).

[0285] In some embodiments, the scFv comprises VL, L1, and VH from N-terminus to C-terminus (VL-L1-VH).

[0286] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:7.

[0287] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:76.

[0288] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:77.

[0289] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:78.

[0290] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:79.

[0291] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:80.

[0292] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:81.

[0293] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:82.

[0294] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:83.

[0295] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:84.

[0296] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:85.

[0297] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:86.

[0298] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:87.

[0299] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:88.

[0300] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:89.

[0301] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:90.

[0302] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:91.

[0303] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:92.

[0304] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:93.

[0305] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:94.

[0306] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:95.

[0307] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:96.

[0308] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:97.

[0309] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:98.

[0310] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:99.

[0311] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:100.

[0312] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:101.

[0313] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:102.

[0314] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:103.

[0315] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:104.

[0316] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:105.

[0317] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:106.

[0318] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO: 107

[0319] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:108.

[0320] Table 1.

[0321]

[0322]

[0323] In a specific embodiment, the L1 comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0324] In some embodiments, the scFv comprises:

[0325] or the HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 164 and the LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 165; or the HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 166 and the LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 167; or the HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO: 168 and the LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 169; or the HCDR1, HCDR2 and HCDR3 of the VH of SEQ ID NO: 204 and the LCDR1, LCDR2 and LCDR3 of the VL of SEQ ID NO: 205; or the HCDR1, HCDR2 and HCDR3 of the VH of SEQ ID NO: 139 and the LCDR1, LCDR2 and LCDR3 of the VL of SEQ ID NO: 140.

[0326] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[0327] SEQ ID NOs: 63, 65, 66, 67, 69, 71, respectively;

[0328] SEQ ID NOs: 63, 65, 66, 68, 70, 71, respectively;

[0329] SEQ ID NOs: 72, 73, 66, 67, 69, 71, respectively;

[0330] SEQ ID NOs: 141, 142, 143, 144, 145 and 146, respectively;

[0331] SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively;

[0332] They are SEQ ID NOs: 176, 177, 178, 179, 180 and 181, respectively.

[0333] SEQ ID NOs: 170, 183, 184, 185, 186 and 187, respectively;

[0334] SEQ ID NOs: 188, 189, 190, 191, 192 and 193, respectively;

[0335] SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively;

[0336] SEQ ID NOs: 147, 148, 143, 144, 145 and 146, respectively;

[0337] SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively;

[0338] SEQ ID NOs: 196, 197, 178, 179, 180, 181, respectively;

[0339] SEQ ID NOs: 198, 199, 184, 185, 186 and 187, respectively;

[0340] SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively; or

[0341] They are SEQ ID NOs: 216, 217, 218, 182, 470 and 209, respectively.

[0342] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively.

[0343] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively.

[0344] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively.

[0345] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively.

[0346] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 182, 470, and 209, respectively.

[0347] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 147, 148, 143, 144, 145, and 146, respectively.

[0348] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively.

[0349] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively.

[0350] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 196, 197, 178, 179, 180, 181, respectively.

[0351] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 198, 199, 184, 185, 186, and 187, respectively.

[0352] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively.

[0353] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 216, 217, 218, 182, 470, and 209, respectively.

[0354] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively.

[0355] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 68, 70, and 71, respectively.

[0356] In some embodiments, the scFv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 72, 73, 66, 67, 69, and 71, respectively.

[0357] In some embodiments, the scFv comprises the VH of SEQ ID NO:137 and the VL of SEQ ID NO:138.

[0358] In some embodiments, the scFv comprises the VH of SEQ ID NO:162 and the VL of SEQ ID NO:163.

[0359] In some embodiments, the scFv comprises the VH of SEQ ID NO:164 and the VL of SEQ ID NO:165.

[0360] In some embodiments, the scFv comprises the VH of SEQ ID NO:166 and the VL of SEQ ID NO:167.

[0361] In some embodiments, the scFv comprises the VH of SEQ ID NO:168 and the VL of SEQ ID NO:169.

[0362] In some embodiments, the scFv comprises the VH of SEQ ID NO:204 and the VL of SEQ ID NO:205.

[0363] In some embodiments, the scFv comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:160.

[0364] In some embodiments, the scFv comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:140.

[0365] In some embodiments, the scFv comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:140.

[0366] In some embodiments, the scFv comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:140.

[0367] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 137 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 138.

[0368] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0369] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 164 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 165.

[0370] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 167.

[0371] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 168 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 169.

[0372] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 204 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 205.

[0373] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 159 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 160.

[0374] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 161 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 140.

[0375] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 139 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 140.

[0376] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 159 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 140.

[0377] In some embodiments, the scFv comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO:162 and a VL of SEQ ID NO:163.

[0378] In some embodiments, the scFv comprises the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0379] In some embodiments, the scFv comprises a VH that is at least 95% identical to the VH of SEQ ID NO:162 and a VL that is at least 95% identical to the VL of SEQ ID NO:163.

[0380] In some embodiments, the scFv comprises a VH that is at least 99% identical to the VH of SEQ ID NO:162 and a VL that is at least 95% identical to the VL of SEQ ID NO:163.

[0381] In some embodiments, the scFv comprises a VH that is at least 99% identical to the VH of SEQ ID NO:162 and a VL that is at least 99% identical to the VL of SEQ ID NO:163.

[0382] In some embodiments, the scFv comprises a VH that is at least 95% identical to the VH of SEQ ID NO:162 and a VL that is at least 99% identical to the VL of SEQ ID NO:163.

[0383] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO: 133, 134, 308, 316, 324, 325, 404, 405, 406, 407, 408, or 409.

[0384] In some embodiments, the scFv comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 133, 134, 308, 316, 324, 325, 404, 405, 406, 407, 408, or 409.

[0385] In some embodiments, the scFv comprises an amino acid sequence that is at least 80% (eg, at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:404.

[0386] In some embodiments, the scFv comprises an amino acid sequence that is at least 80% (eg, at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:405.

[0387] In some embodiments, the scFv comprises the VH of SEQ ID NO:75 and the VL of SEQ ID NO:74.

[0388] In some embodiments, the scFv comprises the VH of SEQ ID NO: 4, 5, or 6 and the VL of SEQ ID NO: 1, 2, or 3.

[0389] In some embodiments, the scFv comprises the VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and the VL of SEQ ID NO: 1, 2, 3, 140, or 160, with the proviso that the antigen binding domain that binds hK2 does not comprise the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0390] In some embodiments, the scFv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:1.

[0391] In some embodiments, the scFv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:2.

[0392] In some embodiments, the scFv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:3.

[0393] In some embodiments, the scFv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:140.

[0394] In some embodiments, the scFv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:160.

[0395] In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:1.

[0396] In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:3.

[0397] In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:140.

[0398] In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:160.

[0399] In some embodiments, the scFv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:1.

[0400] In some embodiments, the scFv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:2.

[0401] In some embodiments, the scFv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:3.

[0402] In some embodiments, the scFv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:140.

[0403] In some embodiments, the scFv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:160.

[0404] In some embodiments, the scFv comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 1.

[0405] In some embodiments, the scFv comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 2.

[0406] In some embodiments, the scFv comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 3.

[0407] In some embodiments, the scFv comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:140.

[0408] In some embodiments, the scFv comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:160.

[0409] In some embodiments, the scFv comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 1.

[0410] In some embodiments, the scFv comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 2.

[0411] In some embodiments, the scFv comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 3.

[0412] In some embodiments, the scFv comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:140.

[0413] In some embodiments, the scFv comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:160.

[0414] In some embodiments, the scFv comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 1.

[0415] In some embodiments, the scFv comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 2.

[0416] In some embodiments, the scFv comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 3.

[0417] In some embodiments, the scFv comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:140.

[0418] In some embodiments, the scFv comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:160.

[0419] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:8.

[0420] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:9.

[0421] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:10.

[0422] In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:11.

[0423] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:12.

[0424] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:13.

[0425] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:14.

[0426] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:15.

[0427] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:16.

[0428] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:17.

[0429] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:18.

[0430] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:19.

[0431] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:20.

[0432] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:21.

[0433] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:22.

[0434] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:23.

[0435] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:135.

[0436] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:136.

[0437] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:318.

[0438] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:319.

[0439] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:320.

[0440] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:321.

[0441] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:322.

[0442] In some embodiments, the scFv comprises the amino acids of SEQ ID NO:323.

[0443] Other antigen-binding domains that bind hK2

[0444] Any of the hK2-binding VH and VL domains identified herein can also be engineered into Fab, F(ab')2, Fd, or Fv formats and their binding to hK2 and thermal stability assessed using the assays described herein.

[0445] In some embodiments, the Fab comprises:

[0446] heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or

[0447] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 162 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 163;

[0448] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 164 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 165;

[0449] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 166 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 167;

[0450] HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 168 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 169; or

[0451] HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 204 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 205.

[0452] In a specific embodiment, the Fab comprises HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO:162 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO:163.

[0453] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[0454] SEQ ID NOs: 63, 65, 66, 67, 69, 71, respectively;

[0455] SEQ ID NOs: 63, 65, 66, 68, 70, 71, respectively;

[0456] SEQ ID NOs: 72, 73, 66, 67, 69, 71, respectively;

[0457] SEQ ID NOs: 141, 142, 143, 144, 145 and 146, respectively;

[0458] SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively;

[0459] SEQ ID NOs: 176, 177, 178, 179, 180 and 181, respectively;

[0460] SEQ ID NOs: 170, 183, 184, 185, 186 and 187, respectively;

[0461] SEQ ID NOs: 188, 189, 190, 191, 192 and 193, respectively;

[0462] SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively;

[0463] SEQ ID NOs: 147, 148, 143, 144, 145 and 146, respectively;

[0464] SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively;

[0465] SEQ ID NOs: 196, 197, 178, 179, 180, 181, respectively;

[0466] SEQ ID NOs: 198, 199, 184, 185, 186 and 187, respectively;

[0467] SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively; or

[0468] They are SEQ ID NOs: 216, 217, 218, 182, 470 and 209, respectively.

[0469] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively.

[0470] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively.

[0471] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively.

[0472] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively.

[0473] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively.

[0474] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 206, 207, 208, 182, 470, 209, respectively.

[0475] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 147, 148, 143, 144, 145, and 146, respectively.

[0476] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively.

[0477] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 196, 197, 178, 179, 180, 181, respectively.

[0478] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 198, 199, 184, 185, 186, and 187, respectively.

[0479] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively.

[0480] In some embodiments, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 216, 217, 218, 182, 470, and 209, respectively.

[0481] In a specific embodiment, the Fab comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, or SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively.

[0482] In some embodiments, the Fab comprises the VH of SEQ ID NO:137 and the VL of SEQ ID NO:138.

[0483] In some embodiments, the Fab comprises the VH of SEQ ID NO:162 and the VL of SEQ ID NO:163.

[0484] In some embodiments, the Fab comprises the VH of SEQ ID NO:164 and the VL of SEQ ID NO:165.

[0485] In some embodiments, the Fab comprises the VH of SEQ ID NO:166 and the VL of SEQ ID NO:167.

[0486] In some embodiments, the Fab comprises the VH of SEQ ID NO:168 and the VL of SEQ ID NO:169.

[0487] In some embodiments, the Fab comprises the VH of SEQ ID NO:204 and the VL of SEQ ID NO:205.

[0488] In some embodiments, the Fab comprises the VH of SEQ ID NO:75 and the VL of SEQ ID NO:74.

[0489] In a specific embodiment, the Fab comprises the VH of SEQ ID NO:162 and the VL of SEQ ID NO:163.

[0490] In some embodiments, the Fab comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0491] In some embodiments, the Fab comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0492] In some embodiments, the Fab comprises the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0493] In some embodiments, the Fab comprises a VH that is at least 95% identical to the VH of SEQ ID NO:162 and a VL that is at least 95% identical to the VL of SEQ ID NO:163.

[0494] In some embodiments, the Fab comprises a VH that is at least 99% identical to the VH of SEQ ID NO:162 and a VL that is at least 95% identical to the VL of SEQ ID NO:163.

[0495] In some embodiments, the Fab comprises a VH that is at least 99% identical to the VH of SEQ ID NO:162 and a VL that is at least 99% identical to the VL of SEQ ID NO:163.

[0496] In some embodiments, the Fab comprises a VH that is at least 99% identical to the VH of SEQ ID NO:162 and a VL that is at least 95% identical to the VL of SEQ ID NO:163.

[0497] In some embodiments, the Fab comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 137 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 138.

[0498] In some embodiments, the Fab comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 164 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 165.

[0499] In some embodiments, the Fab comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 167.

[0500] In some embodiments, the Fab comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 168 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 169.

[0501] In some embodiments, the Fab comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 204 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 205.

[0502] In some embodiments, the Fab comprises a VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and a VL of SEQ ID NO: 1, 2, 3, 140, or 160, with the proviso that the Fab does not comprise a VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 2.

[0503] In some embodiments, the Fab comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:1.

[0504] In some embodiments, the Fab comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:2.

[0505] In some embodiments, the Fab comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:3.

[0506] In some embodiments, the Fab comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:140.

[0507] In some embodiments, the Fab comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:160.

[0508] In some embodiments, the Fab comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:1.

[0509] In some embodiments, the Fab comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:3.

[0510] In some embodiments, the Fab comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:140.

[0511] In some embodiments, the Fab comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:160.

[0512] In some embodiments, the Fab comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:1.

[0513] In some embodiments, the Fab comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:2.

[0514] In some embodiments, the Fab comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:3.

[0515] In some embodiments, the Fab comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:140.

[0516] In some embodiments, the Fab comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:160.

[0517] In some embodiments, the Fab comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 1.

[0518] In some embodiments, the Fab comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 2.

[0519] In some embodiments, the Fab comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 3.

[0520] In some embodiments, the Fab comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:140.

[0521] In some embodiments, the Fab comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:160.

[0522] In some embodiments, the Fab comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 1.

[0523] In some embodiments, the Fab comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 2.

[0524] In some embodiments, the Fab comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 3.

[0525] In some embodiments, the Fab comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:140.

[0526] In some embodiments, the Fab comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:160.

[0527] In some embodiments, the Fab comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 1.

[0528] In some embodiments, the Fab comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 2.

[0529] In some embodiments, the Fab comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 3.

[0530] In some embodiments, the Fab comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:140.

[0531] In some embodiments, the Fab comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:160.

[0532] The VH and VL of a Fab comprising an antigen-binding domain that binds hK2 can be engineered into Fab-Fc HC (VH-CH1-hinge-CH2-CH3) and Fab-Fc LC (VL-CL) formats, respectively. In certain such embodiments, the Fab-Fc HC comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 354. In a specific embodiment, the Fab-Fc HC comprises an amino acid sequence that is identical to SEQ ID NO: 354.

[0533] In some embodiments, the Fab-Fc HC comprises a C-terminal lysine residue (e.g., K477). In some embodiments, the Fab-Fc HC comprises an amino acid sequence having a C-terminal lysine residue and a sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 361. In a specific embodiment, the Fab-Fc HC comprises the amino acid sequence of SEQ ID NO: 361.

[0534] In some embodiments, the Fab-Fc LC comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 221. In a specific embodiment, the Fab-Fc LC comprises an amino acid sequence that is identical to SEQ ID NO: 221.

[0535] As shown in the Examples, particularly suitable antigen binding domains that bind hK2 for incorporation into multispecific constructs comprise a Fab-Fc HC having the amino acid sequence of SEQ ID NO:354 and a Fab-Fc LC having the amino acid sequence of SEQ ID NO:221.

[0536] In some embodiments, F(ab' )2 Include

[0537] heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or

[0538] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 162 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 163;

[0539] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 164 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 165;

[0540] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 166 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 167;

[0541] HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 168 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 169; or

[0542] HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 204 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 205.

[0543] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[0544] SEQ ID NOs: 141, 142, 143, 144, 145 and 146, respectively;

[0545] SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively;

[0546] SEQ ID NOs: 176, 177, 178, 179, 180 and 181, respectively;

[0547] SEQ ID NOs: 170, 183, 184, 185, 186 and 187, respectively;

[0548] SEQ ID NOs: 188, 189, 190, 191, 192 and 193, respectively;

[0549] SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively;

[0550] SEQ ID NOs: 147, 148, 143, 144, 145 and 146, respectively;

[0551] SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively;

[0552] SEQ ID NOs: 196, 197, 178, 179, 180, 181, respectively;

[0553] SEQ ID NOs: 198, 199, 184, 185, 186 and 187, respectively;

[0554] SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively; or

[0555] They are SEQ ID NOs: 216, 217, 218, 182, 470 and 209, respectively.

[0556] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively.

[0557] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively.

[0558] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively.

[0559] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively.

[0560] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively.

[0561] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively.

[0562] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 147, 148, 143, 144, 145, and 146, respectively.

[0563] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively.

[0564] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 196, 197, 178, 179, 180, 181, respectively.

[0565] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 198, 199, 184, 185, 186, and 187, respectively.

[0566] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively.

[0567] In some embodiments, the F(ab')2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 216, 217, 218, 182, 470, and 209, respectively.

[0568] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:137 and the VL of SEQ ID NO:138.

[0569] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:162 and the VL of SEQ ID NO:163.

[0570] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:164 and the VL of SEQ ID NO:165.

[0571] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:166 and the VL of SEQ ID NO:167.

[0572] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:168 and the VL of SEQ ID NO:169.

[0573] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:204 and the VL of SEQ ID NO:205.

[0574] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:75 and the VL of SEQ ID NO:74.

[0575] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and the VL of SEQ ID NO: 1, 2, 3, 140, or 160, with the proviso that the Fab does not comprise the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0576] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:1.

[0577] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:2.

[0578] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:3.

[0579] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:140.

[0580] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:160.

[0581] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:1.

[0582] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:3.

[0583] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:140.

[0584] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:160.

[0585] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:1.

[0586] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:2.

[0587] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:3.

[0588] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:140.

[0589] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:160.

[0590] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 1.

[0591] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 2.

[0592] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 3.

[0593] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:140.

[0594] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:160.

[0595] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 1.

[0596] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 2.

[0597] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 3.

[0598] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:140.

[0599] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:160.

[0600] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 1.

[0601] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 2.

[0602] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 3.

[0603] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:140.

[0604] In some embodiments, the F(ab')2 comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:160.

[0605] In some embodiments, the Fv comprises:

[0606] heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable region (VH) of SEQ ID NO: 137, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of the light chain variable region (VL) of SEQ ID NO: 138; or

[0607] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 162 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 163;

[0608] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 164 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 165;

[0609] HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 166 and LCDR1, LCDR2, and LCDR3 of VL of SEQ ID NO: 167;

[0610] HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 168 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 169; or

[0611] HCDR1, HCDR2 and HCDR3 of VH of SEQ ID NO: 204 and LCDR1, LCDR2 and LCDR3 of VL of SEQ ID NO: 205.

[0612] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[0613] SEQ ID NOs: 141, 142, 143, 144, 145 and 146, respectively;

[0614] SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively;

[0615] SEQ ID NOs: 176, 177, 178, 179, 180 and 181, respectively;

[0616] SEQ ID NOs: 170, 183, 184, 185, 186 and 187, respectively;

[0617] SEQ ID NOs: 188, 189, 190, 191, 192 and 193, respectively;

[0618] SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively;

[0619] SEQ ID NOs: 147, 148, 143, 144, 145 and 146, respectively;

[0620] SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively;

[0621] SEQ ID NOs: 196, 197, 178, 179, 180, 181, respectively;

[0622] SEQ ID NOs: 198, 199, 184, 185, 186 and 187, respectively;

[0623] SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively; or

[0624] They are SEQ ID NOs: 216, 217, 218, 182, 470 and 209, respectively.

[0625] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively.

[0626] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively.

[0627] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively.

[0628] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively.

[0629] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively.

[0630] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively.

[0631] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 147, 148, 143, 144, 145, and 146, respectively.

[0632] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively.

[0633] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 196, 197, 178, 179, 180, 181, respectively.

[0634] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 198, 199, 184, 185, 186, and 187, respectively.

[0635] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively.

[0636] In some embodiments, the Fv comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 216, 217, 218, 182, 470, and 209, respectively.

[0637] In some embodiments, the Fv comprises the VH of SEQ ID NO:137 and the VL of SEQ ID NO:138.

[0638] In some embodiments, the Fv comprises the VH of SEQ ID NO:162 and the VL of SEQ ID NO:163.

[0639] In some embodiments, the Fv comprises the VH of SEQ ID NO:164 and the VL of SEQ ID NO:165.

[0640] In some embodiments, the Fv comprises the VH of SEQ ID NO:166 and the VL of SEQ ID NO:167.

[0641] In some embodiments, the Fv comprises the VH of SEQ ID NO:168 and the VL of SEQ ID NO:169.

[0642] In some embodiments, the Fv comprises the VH of SEQ ID NO:204 and the VL of SEQ ID NO:205.

[0643] In some embodiments, the Fv comprises the VH of SEQ ID NO:75 and the VL of SEQ ID NO:74.

[0644] In some embodiments, the Fv comprises the VH of SEQ ID NO: 4, 5, or 6 and the VL of SEQ ID NO: 1, 2, or 3.

[0645] In some embodiments, the Fv comprises the VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and the VL of SEQ ID NO: 1, 2, 3, 140, or 160, with the proviso that the Fab does not comprise the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[0646] In some embodiments, the Fv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:1.

[0647] In some embodiments, the Fv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:2.

[0648] In some embodiments, the Fv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:3.

[0649] In some embodiments, the Fv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:140.

[0650] In some embodiments, the Fv comprises the VH of SEQ ID NO:4 and the VL of SEQ ID NO:160.

[0651] In some embodiments, the Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:1.

[0652] In some embodiments, the Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:3.

[0653] In some embodiments, the Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:140.

[0654] In some embodiments, the Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:160.

[0655] In some embodiments, the Fv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:1.

[0656] In some embodiments, the Fv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:2.

[0657] In some embodiments, the Fv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:3.

[0658] In some embodiments, the Fv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:140.

[0659] In some embodiments, the Fv comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:160.

[0660] In some embodiments, the Fv comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 1.

[0661] In some embodiments, the Fv comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 2.

[0662] In some embodiments, the Fv comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 3.

[0663] In some embodiments, the Fv comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:140.

[0664] In some embodiments, the Fv comprises the VH of SEQ ID NO:139 and the VL of SEQ ID NO:160.

[0665] In some embodiments, the Fv comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 1.

[0666] In some embodiments, the Fv comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 2.

[0667] In some embodiments, the Fv comprises the VH of SEQ ID NO: 159 and the VL of SEQ ID NO: 3.

[0668] In some embodiments, the Fv comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:140.

[0669] In some embodiments, the Fv comprises the VH of SEQ ID NO:159 and the VL of SEQ ID NO:160.

[0670] In some embodiments, the Fv comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 1.

[0671] In some embodiments, the Fv comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 2.

[0672] In some embodiments, the Fv comprises the VH of SEQ ID NO: 161 and the VL of SEQ ID NO: 3.

[0673] In some embodiments, the Fv comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:140.

[0674] In some embodiments, the Fv comprises the VH of SEQ ID NO:161 and the VL of SEQ ID NO:160.

[0675] In some embodiments, the Fd comprises the VH of SEQ ID NO:75.

[0676] In some embodiments, the Fd comprises the VH of SEQ ID NO:4.

[0677] In some embodiments, the Fd comprises the VH of SEQ ID NO:5.

[0678] In some embodiments, the Fd comprises the VH of SEQ ID NO:6.

[0679] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:210 and the LC of SEQ ID NO:221.

[0680] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:211 and the LC of SEQ ID NO:222.

[0681] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:212 and the LC of SEQ ID NO:223.

[0682] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:213 and the LC of SEQ ID NO:224.

[0683] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:219 and the LC of SEQ ID NO:220.

[0684] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 354 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 221.

[0685] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises a HC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 354 and a LC of SEQ ID NO: 221.

[0686] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO: 354 and a LC that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 221.

[0687] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises a HC that is at least 95% identical to SEQ ID NO:354 and a LC that is at least 95% identical to SEQ ID NO:221.

[0688] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises a HC that is at least 99% identical to SEQ ID NO:354 and a LC that is at least 95% identical to SEQ ID NO:221.

[0689] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises a HC that is at least 99% identical to SEQ ID NO:354 and a LC that is at least 99% identical to SEQ ID NO:221.

[0690] In some embodiments, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises a HC that is at least 95% identical to SEQ ID NO:354 and a LC that is at least 99% identical to SEQ ID NO:221.

[0691] In a specific embodiment, the isolated anti-hK2 antibody or antigen-binding fragment thereof comprises the HC of SEQ ID NO:354 and the LC of SEQ ID NO:221.

[0692] Homologous antigen-binding domains and antigen-binding domains with conservative substitutions

[0693] Variants of hK2-binding antigen-binding domains are within the scope of the present disclosure. For example, a variant may comprise 1, 2, 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, or 29 amino acid substitutions in the hK2-binding antigen-binding domain, as long as they retain or have improved functional properties compared to the parent antigen-binding domain. In some embodiments, the sequence identity to the hK2-binding antigen-binding domain of the present disclosure may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the variation is in the framework region. In some embodiments, variants are generated by conservative substitutions.

[0694] For example, an antigen binding domain that binds hK2 can comprise substitutions at residue positions D16, A23, P41, G45, I49, M70, A88, and V89 in VH (residue numbering according to hu11B6_VH of SEQ ID NO: 5) and residue positions D1, D9, S10, A12, V13, L15, I21, N22, K24, K49, R58, V62, D64, S80, L82, Q83, A84, V87, A88, Y91, Q104, and L108 in VL (residue numbering according to hu11B6_VL of SEQ ID NO: 2). Conservative substitutions can be made at any given position, and the desired characteristics of the resulting variant antigen binding domain that binds hK2 are tested in the assays described herein.

[0695] In some embodiments, the isolated protein comprising an antigen binding domain that binds hK2 comprises a VH and a VL that are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH and VL, respectively, of the antigen binding domain that binds hK2 disclosed herein.

[0696] Also provided are antigen binding domains that bind hK2 comprising a VH and a VL that are at least 80% identical to:

[0697] VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138;

[0698] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140;

[0699] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1;

[0700] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2;

[0701] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3;

[0702] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1;

[0703] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3;

[0704] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1;

[0705] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2;

[0706] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3;

[0707] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160;

[0708] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140;

[0709] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140;

[0710] VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163;

[0711] VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165;

[0712] VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167;

[0713] VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169; or

[0714] The VH of SEQ ID NO: 204 and the VL of SEQ ID NO: 205.

[0715] In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%. In some embodiments, the identity is 94%. In some embodiments, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

[0716] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 137 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 138.

[0717] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0718] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 164 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 165.

[0719] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 167.

[0720] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 166 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 444.

[0721] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 168 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 169.

[0722] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 204 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 205.

[0723] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 85% identical to the VH of SEQ ID NO:162 and a VL of SEQ ID NO:163.

[0724] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 90% identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0725] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 91% identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0726] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 92% identical to the VH of SEQ ID NO:162 and a VL of SEQ ID NO:163.

[0727] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 93% identical to the VH of SEQ ID NO:162 and a VL of SEQ ID NO:163.

[0728] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 94% identical to the VH of SEQ ID NO:162 and a VL of SEQ ID NO:163.

[0729] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 95% identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0730] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 96% identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0731] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 97% identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0732] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 98% identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0733] In some embodiments, the antigen binding domain that binds hK2 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0734] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 85% identical to the VL of SEQ ID NO: 163.

[0735] In some embodiments, the antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 162 and a VL that is at least 90% identical to a VL of SEQ ID NO: 163.

[0736] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 91% identical to the VL of SEQ ID NO: 163.

[0737] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 92% identical to the VL of SEQ ID NO: 163.

[0738] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 93% identical to the VL of SEQ ID NO: 163.

[0739] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 94% identical to the VL of SEQ ID NO: 163.

[0740] In some embodiments, the antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 162 and a VL that is at least 95% identical to a VL of SEQ ID NO: 163.

[0741] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 96% identical to the VL of SEQ ID NO: 163.

[0742] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 97% identical to the VL of SEQ ID NO: 163.

[0743] In some embodiments, the antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 98% identical to the VL of SEQ ID NO: 163.

[0744] In some embodiments, the antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 162 and a VL that is at least 99% identical to a VL of SEQ ID NO: 163.

[0745] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap, which parameters need to be introduced for optimal alignment of the two sequences.

[0746] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch (J Mol Biol 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0747] In some embodiments, the variant antigen binding domain that binds hK2 comprises one or two conservative substitutions in any CDR region while retaining the desired functional properties of the parent antigen binding fragment that binds hK2.

[0748] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modification. Conservative modifications include amino acid substitutions, additions, and deletions. "Conservative amino acid substitutions" are substitutions in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well-defined and include amino acids having the following side chains: acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cysteine, methionine). In addition, any native residue in the polypeptide can also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643: 55-67; ​​Sasaki et al., (1988) Adv Biophys 35: 1-24). Amino acid substitutions for the antibodies of the invention can be made by known methods, for example, by PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, a library of variants can be generated, for example, using random (NNK) or non-random codons (e.g., DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The characteristics of the resulting variants can be tested using the assays described herein.

[0749] Methods for generating antigen-binding fragments that bind hK2

[0750] The antigen-binding domains that bind hK2 provided herein can be generated using a variety of techniques. For example, the hybridoma method of Kohler and Milstein can be used to identify VH / VL pairs that bind hK2. In the hybridoma method, mice or other host animals (such as hamsters, rats, or chickens) are immunized with human and / or cyno hK2, and spleen cells from the immunized animal are then fused with myeloma cells using standard methods to form hybridoma cells. Colonies generated from single immortalized hybridoma cells can be screened for the production of antibodies containing antigen-binding domains that bind hK2 with desired properties, such as binding specificity, cross-reactivity or lack of binding specificity, lack of cross-reactivity, affinity for the antigen, and any desired functionality.

[0751] Antigen binding domains that bind hK2 and are generated by immunizing non-human animals can be humanized. Exemplary humanization techniques that include selecting a human acceptor framework include CDR grafting (U.S. Patent No. 5,225,539), SDR grafting (U.S. Patent No. 6,818,749), resurfacing (Padlan, (1991) Mol Immunol 28: 489-499), resurfacing of specificity-determining residues (U.S. Patent Publication No. 2010 / 0261620), human framework reshaping (U.S. Patent No. 8,748,356), or superhumanization (U.S. Patent No. 7,709,226). In these methods, the CDRs or a subset of CDR residues of a parent antibody are transferred to a human framework, which can be selected based on its overall homology to the parent framework, based on similarity in CDR length or canonical structural identity, or a combination thereof.

[0752] The humanized antigen binding domain can be further optimized to improve its selectivity or affinity for the desired antigen by incorporating altered framework support residues to maintain binding affinity (back mutations) using techniques such as those described in International Patent Publication Nos. WO1090 / 007861 and WO1992 / 22653, or by introducing variations in any CDR to improve the affinity of the antigen binding domain.

[0753] Transgenic animals (such as mice, rats, or chickens) carrying human immunoglobulin (Ig) loci in their genomes can be used to generate antigen-binding fragments that bind hK2 and are described, for example, in U.S. Patent No. 6,150,584, International Patent Publication No. WO1999 / 45962, International Patent Publication Nos. WO2002 / 066630, WO2002 / 43478, WO2002 / 043478, and WO1990 / 04036. The endogenous immunoglobulin loci in such animals can be disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the genome of the animal by homologous or nonhomologous recombination using a transchromosome or minigene. Companies such as Regeneron (http: / / www.regeneron.com), Harbour Antibodies (http: / / www.harbourantibodies.com), Open Monoclonal Technology, Inc. (OMT) (http: / / www.omtinc.net), KyMab (http: / / www.kymab.com), Trianni (http: / / www.trianni.com), and Ablexis (http: / / www.ablexis.com) can be invited to use the above technology to provide human antibodies against selected antigens. In some embodiments, Ablexis mice and OmniRats rats are immunized with soluble full-length KLK2 protein (human kallikrein-2 6-His protein) (SEQ ID NO: 454).

[0754] Antigen binding domains that bind hK2 can be selected from phage display libraries in which phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single-chain antibodies (scFv), or unpaired or paired antibody variable regions. Antigen binding domains that bind hK2 can be isolated, for example, from phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins using the phage pIX coat protein, as described in Shi et al. (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO09 / 085462. The library can be screened for phage binding to human and / or cyno hK2, and the resulting positive clones can be further characterized, and Fabs can be isolated from clone lysates and converted to scFv or other configurations of antigen binding fragments.

[0755] Preparation of immunogenic antigens and generation of antigen-binding domains of the present disclosure can be performed using any suitable technique, such as recombinant protein production. Immunogenic antigens can be administered to animals in the form of purified proteins or protein mixtures (including whole cells or cell extracts or tissue extracts), or the antigens can be generated de novo in the animal from nucleic acids encoding the antigen or a portion thereof.

[0756] Conjugated to a half-life extending moiety

[0757] The hK2-binding antigen binding domains disclosed herein can be conjugated to a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin binding proteins and / or domains, transferrin and fragments and analogs thereof, immunoglobulins (Ig) or fragments thereof (such as Fc regions). The amino acid sequences of the aforementioned half-life extending moieties are known. Ig or fragments thereof include all isotypes, i.e., IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0758] Additional half-life extending moieties that can be conjugated to the hK2-binding antigen-binding domains of the present disclosure include, for example, polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000; fatty acids and fatty acid esters of varying chain lengths, such as laurate, myristate, stearate, arachidate, behenate, oleate, arachidonic acid, suberic acid, tetradecanedioic acid, octadecanedioic acid, docosandioic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) to achieve the desired properties. These moieties can be directly fused to the hK2-binding antigen-binding domains of the present disclosure and can be produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods can be used to attach the moieties to the recombinantly produced hK2-binding antigen-binding domains of the present disclosure.

[0759] The polyethylene glycol moiety can be conjugated to the hK2-binding antigen binding domain of the present disclosure, for example, by incorporating a cysteine ​​residue into the C-terminus of the hK2-binding antigen binding domain of the present disclosure or engineering a cysteine ​​into a residue position facing away from the hK2 binding site and attaching a polyethylene glycol group to the cysteine ​​using well-known methods.

[0760] In some embodiments, the antigen binding fragment that binds hK2 is conjugated to a half-life extending moiety.

[0761] In some embodiments, the half-life extending moiety is an immunoglobulin (Ig), a fragment of said Ig, an Ig constant region, a fragment of said Ig constant region, an Fc region, transferrin, albumin, an albumin binding domain, or polyethylene glycol. In some embodiments, the half-life extending moiety is an Ig constant region.

[0762] In some embodiments, the half-life extending moiety is Ig.

[0763] In some embodiments, the half-life extending moiety is a fragment of Ig.

[0764] In some embodiments, the half-life extending moiety is an Ig constant region.

[0765] In some embodiments, the half-life extending moiety is a fragment of an Ig constant region.

[0766] In some embodiments, the half-life extending moiety is an Fc region.

[0767] In some embodiments, the half-life extending moiety is albumin.

[0768] In some embodiments, the half-life extending moiety is an albumin binding domain.

[0769] In some embodiments, the half-life extending moiety is transferrin.

[0770] In some embodiments, the half-life extending moiety is polyethylene glycol.

[0771] Known in vivo models can be used to evaluate the pharmacokinetic properties of the hK2 binding antigen binding domain conjugated to a half-life extending moiety.

[0772] Conjugated to immunoglobulin (Ig) constant regions or fragments of Ig constant regions

[0773] The hK2-binding antigen-binding domains of the present disclosure can be conjugated to Ig constant regions or fragments of Ig constant regions to confer antibody-like properties, including Fc effector functions, i.e., C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis or downregulation of cell surface receptors (e.g., B cell receptor; BCR). The Ig constant region or fragment of the Ig constant region also acts as a half-life extending moiety as discussed herein. The hK2-binding antigen-binding domains of the present disclosure can be engineered into conventional full-length antibodies using standard methods. Full-length antibodies comprising an hK2-binding antigen-binding domain can be further engineered as described herein.

[0774] The immunoglobulin heavy chain constant region is composed of the subdomains C1, hinge, C2, and C3. The C1 domain spans residues A118-V215 on the heavy chain, C2 domain residues A231-K340, and C3 domain residues G341-K447, with residues numbered according to the EU index. In some cases, G341 is referred to as the C2 domain residue. The hinge is generally defined as including E216 and ending at P230 in human IgG1. The Ig Fc region comprises at least the C2 and C3 domains of the Ig constant region, and therefore comprises at least the region from about A231 to K447 of the Ig heavy chain constant region.

[0775] The present invention also provides an hK2-binding antigen binding domain conjugated to an immunoglobulin (Ig) constant region or a fragment of an Ig constant region.

[0776] In some embodiments, the Ig constant region is a heavy chain constant region.

[0777] In some embodiments, the Ig constant region is a light chain constant region.

[0778] In some embodiments, the fragment of the Ig constant region comprises an Fc region.

[0779] In some embodiments, the fragment of the Ig constant region comprises a CH2 domain.

[0780] In some embodiments, the fragment of the Ig constant region comprises a CH3 domain.

[0781] In some embodiments, the fragment of the Ig constant region comprises a CH2 domain and a CH3 domain.

[0782] In some embodiments, the fragment of the Ig constant region comprises at least a portion of the hinge, the CH2 domain, and the CH3 domain. The portion of the hinge refers to one or more amino acid residues of the Ig hinge.

[0783] In some embodiments, the fragment of the Ig constant region comprises a hinge, a CH2 domain, and a CH3 domain.

[0784] In some embodiments, the antigen binding domain that binds hK2 is conjugated to the N-terminus of an Ig constant region or a fragment of an Ig constant region.

[0785] In some embodiments, the antigen binding domain that binds hK2 is conjugated to the C-terminus of an Ig constant region or a fragment of an Ig constant region.

[0786] In some embodiments, the antigen binding domain that binds hK2 is conjugated to an Ig constant region or a fragment of an Ig constant region via a second linker (L2).

[0787] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO: 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

[0788] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:7.

[0789] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:76.

[0790] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:77.

[0791] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:78.

[0792] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:79.

[0793] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:80.

[0794] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:81.

[0795] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:82.

[0796] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:83.

[0797] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:84.

[0798] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:85.

[0799] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:86.

[0800] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:87.

[0801] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:88.

[0802] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:89.

[0803] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:90.

[0804] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:91.

[0805] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:92.

[0806] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:93.

[0807] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:94.

[0808] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:95.

[0809] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:96.

[0810] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:97.

[0811] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:98.

[0812] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:99.

[0813] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:100.

[0814] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:101.

[0815] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:102.

[0816] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:103.

[0817] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:104.

[0818] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:105.

[0819] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:106.

[0820] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:107.

[0821] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO:108.

[0822] The hK2-binding antigen binding domains of the present disclosure conjugated to an Ig constant region or fragment of an Ig constant region can be evaluated using several known assays. Binding to hK2 can be assessed using the methods described herein. The altered properties conferred by an Ig constant domain or fragment of an Ig constant region (such as an Fc region) can be determined in Fc receptor binding assays using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII, or FcRn receptors, or using cell-based assays that measure, for example, ADCC, CDC, or ADCP.

[0823] ADCC can be assessed using an in vitro assay using cells expressing hK2 as target cells and NK cells as effector cells. Cell lysis is detected based on the release of markers (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) from the lysed cells. In an exemplary assay, target cells can be used at a ratio of 1 target cell to 4 effector cells. Target cells are pre-labeled with BATDA and mixed with effector cells and test antibodies. The samples are incubated for 2 hours, and the cell lysis rate is measured by measuring the BATDA released into the supernatant. The data are normalized to the maximum cytotoxicity using 0.67% Triton X-100 (Sigma Aldrich) and the minimum control determined by spontaneous release of BATDA from target cells in the absence of any antibody.

[0824] ADCP can be assessed by using monocyte-derived macrophages as effector cells and any hK2-expressing cells as target cells, the cells being engineered to express GFP or other marker molecules. In an exemplary assay, the effector cell: target cell ratio can be, for example, 4:1. The effector cells can be incubated with the target cells for 4 hours with or without the antibodies of the invention. After incubation, the cells can be separated using accutase. Macrophages can be identified using anti-CD11b antibodies and anti-CD14 antibodies coupled to fluorescent markers, and standard methods can be used to identify macrophages based on CD11 + CD14 + The percentage of phagocytosis was determined by the % GFP fluorescence in macrophages.

[0825] Cellular CDC can be measured, for example, by: Daudi cells are plated at 1×10 5 Cells / well (50 μL / well) were seeded into RPMI-B (RPMI supplemented with 1% BSA), 50 μL of the test protein was added to the well at a final concentration between 0-100 μg / mL, the reaction was incubated at room temperature for 15 minutes, 11 μL of pooled human serum was added to the well, and the reaction was incubated for 45 minutes at 37° C. The % of propidium iodide-stained cells can be detected in a FACS assay as the percentage (%) of lysed cells using standard methods.

[0826] In some embodiments, the antigen-binding domain that binds hK2 is conjugated to an IgG1 heavy chain constant region or a fragment of an IgG1 heavy chain constant region. In some embodiments, the antigen-binding domain that binds hK2 is conjugated to an IgG1 heavy chain constant region or a fragment of an IgG1 heavy chain constant region (e.g., comprising hinge-CH2-CH3), wherein the IgG1 heavy chain constant region comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 110. In some embodiments, the IgG1 heavy chain constant region comprises Fc silent mutations (L234A_L235A_D265S) and T350V_T366L_K392L_T394W mutations designed to promote selective heterodimerization. In some embodiments, the antigen-binding domain that binds hK2 is conjugated to an IgG1 heavy chain constant region or a fragment of an IgG1 heavy chain constant region, wherein the IgG1 heavy chain constant region has the amino acid sequence of SEQ ID NO: 110.

[0827] In some embodiments, the antigen binding domain that binds hK2 is conjugated to an IgG1 heavy chain constant region (e.g., CH1-hinge-CH2-CH3) comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 378. In a specific embodiment, the antigen binding domain that binds hK2 is conjugated to an IgG1 heavy chain constant region, or a fragment of an IgG1 heavy chain constant region, comprising the amino acid sequence of SEQ ID NO: 378.

[0828] In some embodiments, the antigen binding domain that binds hK2 is conjugated to an IgG1 light chain constant region comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 309. In some embodiments, the antigen binding domain that binds hK2 is conjugated to an IgG1 light chain constant region comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO: 447. In some embodiments, the antigen binding domain that binds hK2 is conjugated to an IgG1 light chain constant region comprising an amino acid sequence that is identical to SEQ ID NO: 309 or 447. In a specific embodiment, the antigen binding domain that binds hK2 is conjugated to an IgG1 light chain constant region comprising an amino acid sequence that is identical to SEQ ID NO: 309.

[0829] In a specific embodiment, the isolated protein disclosed herein comprises: an antigen binding domain that binds hK2 conjugated to an IgG1 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 378, and an antigen binding domain that binds hK2 conjugated to an IgG1 light chain constant region comprising the amino acid sequence identical to SEQ ID NO: 309.

[0830] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378.

[0831] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and a first Ig light chain constant region or a fragment of a first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309.

[0832] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 309.

[0833] In a specific embodiment, the isolated protein disclosed herein comprises a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3) and a second Ig constant region or a fragment of a second Ig constant region comprising an amino acid sequence identical to SEQ ID NO: 109.

[0834] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 309, and wherein the isolated protein further comprises a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), and a second Ig constant region, or a fragment of a second Ig constant region, comprising an amino acid sequence identical to SEQ ID NO: 109.

[0835] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region, wherein the first Ig heavy chain constant region comprises an amino acid sequence identical to SEQ ID NO: 378.

[0836] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and a first Ig light chain constant region or a fragment of a first Ig light chain constant region, wherein the first Ig light chain constant region comprises an amino acid sequence identical to SEQ ID NO: 309.

[0837] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, said first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, said first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309.

[0838] In a specific embodiment, the isolated protein disclosed herein comprises: a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261; and a second Ig constant region or a fragment of a second Ig constant region, wherein the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO: 109.

[0839] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, said first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, said first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309, and wherein the isolated protein further comprises: a second antigen binding domain that binds a lymphocyte antigen, such as CD3, wherein the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261; and a second Ig constant region or a fragment of a second Ig constant region, wherein the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO: 109.

[0840] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region, wherein the first Ig heavy chain constant region comprises an amino acid sequence identical to SEQ ID NO: 378.

[0841] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and a first Ig light chain constant region or a fragment of a first Ig light chain constant region, wherein the first Ig light chain constant region comprises an amino acid sequence identical to SEQ ID NO: 309.

[0842] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region, said first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region or a fragment of a first Ig light chain constant region, said first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309.

[0843] In a specific embodiment, the isolated protein disclosed herein comprises: a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the second antigen binding domain that binds a lymphocyte antigen comprises the amino acid sequence of SEQ ID NO: 331; and a second Ig constant region or a fragment of a second Ig constant region, wherein the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO: 109.

[0844] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, the first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, the first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309, and wherein the isolated protein further comprises a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the second antigen binding domain that binds a lymphocyte antigen comprises the amino acid sequence of SEQ ID NO: 331 and a second Ig constant region, or a fragment of a second Ig constant region, the second Ig constant region comprising an amino acid sequence identical to SEQ ID NO: 109.

[0845] Proteins comprising the hK2-binding antigen-binding domain of the present disclosure

[0846] The hK2-binding antigen binding domains of the present disclosure can be engineered into monospecific or multispecific proteins of various designs using standard methods.

[0847] The present disclosure also provides a monospecific protein comprising the hK2-binding antigen-binding domain of the present disclosure.

[0848] In some embodiments, the monospecific protein is an antibody.

[0849] The present disclosure also provides a multispecific protein comprising the hK2-binding antigen-binding domain of the present disclosure.

[0850] In some embodiments, the multispecific protein is bispecific.

[0851] In some embodiments, the multispecific protein is trispecific.

[0852] In some embodiments, the multispecific protein is tetraspecific.

[0853] In some embodiments, the multispecific protein is monovalent for binding to hK2.

[0854] In some embodiments, the multispecific protein is bivalent with respect to binding to hK2.

[0855] The present disclosure also provides an isolated multispecific protein comprising a first antigen binding domain that binds hK2 and a second antigen binding domain that binds a lymphocyte antigen, such as CD3.

[0856] In some embodiments, the lymphocyte antigen is a T cell antigen.

[0857] In some embodiments, the T cell antigen is CD8 + T cell antigens.

[0858] In some embodiments, the lymphocyte antigen is a NK cell antigen.

[0859] In some embodiments, the lymphocyte antigen is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

[0860] In some embodiments, the lymphocyte antigen is CD3ε.

[0861] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[0862] In some embodiments, the anti-hK2 / anti-CD3 protein is bispecific.

[0863] In some embodiments, the first antigen binding domain that binds hK2 and / or the second antigen binding domain that binds a lymphocyte antigen comprises scFv, (scFv)2, Fv, Fab, F(ab')2, Fd, dAb, or VHH.

[0864] In some embodiments, the first antigen binding domain that binds hK2 and / or the second antigen binding domain that binds a lymphocyte antigen comprises a Fab.

[0865] In certain embodiments, the first antigen binding domain that binds hK2 comprises a Fab.

[0866] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises a Fab.

[0867] In some embodiments, the first antigen binding domain that binds hK2 and / or the second antigen binding domain that binds a lymphocyte antigen comprises F(ab')2.

[0868] In some embodiments, the first antigen binding domain that binds hK2 comprises F(ab')2.

[0869] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises F(ab')2.

[0870] In some embodiments, the first antigen binding domain that binds hK2 and / or the second antigen binding domain that binds a lymphocyte antigen comprises a VHH.

[0871] In some embodiments, the first antigen binding domain that binds hK2 comprises a VHH.

[0872] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises a VHH.

[0873] In some embodiments, the first antigen binding domain that binds hK2 and / or the second antigen binding domain that binds a lymphocyte antigen comprises an Fv.

[0874] In some embodiments, the first antigen binding domain that binds hK2 comprises an Fv.

[0875] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises an Fv.

[0876] In some embodiments, the first antigen binding domain that binds hK2 and / or the second antigen binding domain that binds a lymphocyte antigen comprises Fd.

[0877] In some embodiments, the first antigen binding domain that binds hK2 comprises Fd.

[0878] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises Fd.

[0879] In some embodiments, the first antigen binding domain that binds hK2 and / or the second antigen binding domain that binds a lymphocyte antigen comprises a scFv.

[0880] In a particular embodiment, the multispecific protein is bispecific, wherein the first antigen binding domain that binds hK2 comprises a Fab and the second antigen binding domain that binds a lymphocyte antigen (eg, CD3) comprises a scFv.

[0881] In some embodiments, the bound first antigen binding domain comprises a scFv.

[0882] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises a scFv.

[0883] In some embodiments, the first antigen binding domain that binds hK2 comprises a scFv and the second antigen binding domain that binds a lymphocyte antigen comprises a Fab.

[0884] In a specific embodiment, the first antigen binding domain that binds hK2 comprises a Fab and the second antigen binding domain that binds a lymphocyte antigen comprises a scFv.

[0885] In some embodiments, the first antigen binding domain that binds hK2 comprises a scFv and the second antigen binding domain that binds a lymphocyte antigen comprises a Fab'.

[0886] In some embodiments, the first antigen binding domain that binds hK2 comprises a Fab' and the second antigen binding domain that binds a lymphocyte antigen comprises a scFv.

[0887] In some embodiments, the first antigen binding domain that binds hK2 comprises a scFv and the second antigen binding domain that binds a lymphocyte antigen comprises an Fv.

[0888] In some embodiments, the first antigen binding domain that binds hK2 comprises a dAb and the second antigen binding domain that binds a lymphocyte antigen comprises a scFv.

[0889] In some embodiments, the first antigen binding domain that binds hK2 comprises a scFv and the second antigen binding domain that binds a lymphocyte antigen comprises a dAb.

[0890] In some embodiments, the first antigen binding domain that binds hK2 comprises an Fd and the second antigen binding domain that binds a lymphocyte antigen comprises an scFv.

[0891] In some embodiments, the first antigen binding domain that binds hK2 comprises a scFv and the second antigen binding domain that binds a lymphocyte antigen comprises a VHH.

[0892] In some embodiments, the first antigen binding domain that binds hK2 comprises a VHH and the second antigen binding domain that binds a lymphocyte antigen comprises a scFv.

[0893] In some embodiments, the first antigen binding domain that binds hK2 comprises an Fv and the second antigen binding domain that binds a lymphocyte antigen comprises an scFv.

[0894] In some embodiments, the first antigen binding domain that binds hK2 comprises a scFv and the second antigen binding domain that binds a lymphocyte antigen comprises an Fd.

[0895] In some embodiments, the scFv comprises, from N-terminus to C-terminus, VH, a first linker (L1), and VL (VH-L1-VL) or the VL, the L1, and the VH (VL-L1-VH).

[0896] In some embodiments, the L1 comprises about 5-50 amino acids.

[0897] In some embodiments, the L1 comprises about 5-40 amino acids.

[0898] In some embodiments, the L1 comprises about 10-30 amino acids.

[0899] In some embodiments, the L1 comprises about 10-20 amino acids.

[0900] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO: 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

[0901] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:7.

[0902] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:76.

[0903] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:77.

[0904] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:78.

[0905] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:79.

[0906] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:80.

[0907] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:81.

[0908] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:82.

[0909] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:83.

[0910] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:84.

[0911] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:85.

[0912] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:86.

[0913] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:87.

[0914] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:88.

[0915] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:89.

[0916] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:90.

[0917] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:91.

[0918] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:92.

[0919] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:93.

[0920] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:94.

[0921] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:95.

[0922] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:96.

[0923] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:97.

[0924] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:98.

[0925] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:99.

[0926] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:100.

[0927] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:101.

[0928] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:102.

[0929] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:103.

[0930] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:104.

[0931] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:105.

[0932] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:106.

[0933] In some embodiments, the L1 comprises the amino acid of SEQ ID NO:107.

[0934] In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:108.

[0935] In a specific embodiment, the L1 comprises the amino acid sequence of SEQ ID NO:7.

[0936] In some embodiments, the first antigen binding domain that binds hK2 comprises a HCDR1 of SEQ ID NO: 63, 72, 141, 147, 170, 176, 188, 194, 196, 198, 200, 206, or 216, a HCDR2 of SEQ ID NO: 64, 65, 73, 142, 148, 171, 177, 188, 189, 195, 197, 199, 201, 207, or 217, a HCDR3 of SEQ ID NO: 66, 143, 172, 178, 184, 190, 208, or 218, a LCDR1 of SEQ ID NO: 67, 68, 144, 173, 179, 182, 185, or 191, a SE0 LCDR2 of SEQ ID NO: 69, 70, 145, 174, 180, 186, 192 or 470, and LCDR3 of SEQ ID NO: 71, 146, 175, 181, 187, 193 or 209.

[0937] In some embodiments, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[0938] SEQ ID NOs: 63, 65, 66, 68, 70 and 71, respectively;

[0939] SEQ ID NOs: 63, 64, 66, 67, 69 and 71, respectively;

[0940] SEQ ID NOs: 63, 65, 66, 67, 69 and 71, respectively;

[0941] SEQ ID NOs: 141, 142, 143, 144, 145 and 146, respectively;

[0942] SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively;

[0943] SEQ ID NOs: 176, 177, 178, 179, 180 and 181, respectively;

[0944] SEQ ID NOs: 170, 183, 184, 185, 186 and 187, respectively;

[0945] SEQ ID NOs: 188, 189, 190, 191, 192 and 193, respectively;

[0946] SEQ ID NOs: 206, 207, 208, 182, 470, and 209, respectively;

[0947] SEQ ID NOs: 147, 148, 143, 144, 145 and 146, respectively;

[0948] SEQ ID NOs: 72, 73, 66, 68, 70 and 71, respectively;

[0949] SEQ ID NOs: 72, 73, 66, 67, 69 and 71, respectively;

[0950] SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively;

[0951] SEQ ID NOs: 196, 197, 178, 179, 190 and 181, respectively;

[0952] SEQ ID NOs: 198, 199, 184, 185, 186 and 187, respectively;

[0953] SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively; or

[0954] They are SEQ ID NOs: 216, 217, 218, 182, 470 and 209, respectively.

[0955] In a specific embodiment, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, or SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively.

[0956] In a specific embodiment, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second antigen binding domain binds a lymphocyte antigen, which is optionally CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

[0957] In a specific embodiment, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively, and the second antigen binding domain binds a lymphocyte antigen, which is optionally CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

[0958] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:137 and the VL of SEQ ID NO:138.

[0959] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:162 and the VL of SEQ ID NO:163.

[0960] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:164 and the VL of SEQ ID NO:165.

[0961] In some embodiments, the first antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 166 and a VL of SEQ ID NO: 167.

[0962] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:168 and the VL of SEQ ID NO:169.

[0963] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:204 and the VL of SEQ ID NO:205.

[0964] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:75 and the VL of SEQ ID NO:74.

[0965] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:75 and the VL of SEQ ID NO:74.

[0966] In some embodiments, the first antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0967] In some embodiments, the first antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[0968] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[0969] In some embodiments, the first antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163, and the second antigen binding domain binds a lymphocyte antigen, optionally CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

[0970] In some embodiments, the first antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163, and the second antigen binding domain binds a lymphocyte antigen, optionally CD3, CD3 epsilon (CD3 epsilon), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

[0971] In some embodiments, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163, and the second antigen binding domain binds a lymphocyte antigen, optionally CD3, CD3 epsilon (CD3 epsilon), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

[0972] In a specific embodiment, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO:162 and the VL of SEQ ID NO:163.

[0973] In a specific embodiment, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the second antigen binding domain binds a lymphocyte antigen, which is optionally CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

[0974] In some embodiments, the first antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 4, 5, 6, 139, 159, or 161 and a VL of SEQ ID NO: 1, 2, 3, 140, or 160, with the proviso that the Fab does not comprise a VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 2.

[0975] In some embodiments, the first antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 4, 5, or 6 and a VL of SEQ ID NO: 1, 2, or 3, with the proviso that the first antigen binding domain that binds hK2 does not comprise both a VH of SEQ ID NO: 5 and a VL of SEQ ID NO: 2.

[0976] In some embodiments, the first antigen binding domain that binds hK2 comprises:

[0977] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1;

[0978] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2;

[0979] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3;

[0980] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140;

[0981] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160;

[0982] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1;

[0983] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3;

[0984] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140;

[0985] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160;

[0986] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1;

[0987] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2;

[0988] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3;

[0989] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140;

[0990] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160;

[0991] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1;

[0992] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2;

[0993] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3;

[0994] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140;

[0995] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 160;

[0996] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1;

[0997] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2;

[0998] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3;

[0999] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140;

[1000] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160;

[1001] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1;

[1002] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2;

[1003] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3;

[1004] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or

[1005] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 160.

[1006] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 133, 134, 135, 136, 308, 316, 318, 319, 320, 321, 322, 323, 324, 325, 404, 405, 406, 407, 408, or 409.

[1007] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:8.

[1008] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:9.

[1009] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:10.

[1010] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:11.

[1011] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:12.

[1012] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:13.

[1013] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:14.

[1014] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:15.

[1015] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:16.

[1016] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:17.

[1017] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:18.

[1018] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:19.

[1019] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:20.

[1020] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:21.

[1021] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:22.

[1022] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:23.

[1023] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:133.

[1024] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:134.

[1025] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:135.

[1026] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:136.

[1027] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:308.

[1028] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:316.

[1029] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:318.

[1030] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:319.

[1031] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:320.

[1032] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:321.

[1033] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:322.

[1034] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:323.

[1035] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:324.

[1036] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:325.

[1037] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:404.

[1038] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:405.

[1039] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:406.

[1040] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:407.

[1041] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:408.

[1042] In some embodiments, the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO:409.

[1043] In some embodiments, the first antigen binding domain that binds hK2 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO: 404.

[1044] In some embodiments, the first antigen binding domain that binds hK2 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:405.

[1045] In a specific embodiment, the first antigen-binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO: 404 or 405. The present disclosure also provides a second binding domain that binds a lymphocyte antigen, wherein the lymphocyte-binding antigen-binding domain comprises the heavy chain variable region (VH) of SEQ ID NO: 248 and the light chain variable region (VL) of SEQ ID NO: 157. SEQ ID NO: 248 and SEQ ID NO: 157 represent the VH and VL amino acid sequences of the parent antibody CD3B815 antibody.

[1046] VL consensus sequence (SQ ID NO: 157)

[1047] DIQX1TQSPX2X3LSX4SX5GX6RVX7X8X9CRARQSIGTAIHWYQQKX 10 X 11 X 12 X 13 PX 14 LLIX15 YASESISGX 16 PSRFSGSGSGTDFTLTIX 17 SX 18 QX 19 EDX 20 AX 21 YYCQQSX 22 SWPYTFGX 23 GTKLEIK

[1048] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises: HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 260; or

[1049] HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[1050] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises:

[1051] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 249; or

[1052] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 250; or

[1053] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251; or

[1054] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 252; or

[1055] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 253; or

[1056] The VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 254.

[1057] In some embodiments, the second binding domain that binds a lymphocyte antigen comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 248 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 251.

[1058] In some embodiments, the second binding domain that binds a lymphocyte antigen comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 248 and a VL of SEQ ID NO: 251.

[1059] In some embodiments, the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 251.

[1060] In a specific embodiment, the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO:248 and the VL of SEQ ID NO:251.

[1061] In some embodiments, the second antigen binding domain that binds a lymphocyte antigen comprises: HCDR1 of SEQ ID NO: 116, HCDR2 of SEQ ID NO: 117, HCDR3 of SEQ ID NO: 118, LCDR1 of SEQ ID NO: 119, LCDR2 of SEQ ID NO: 120, and LCDR3 of SEQ ID NO: 121; or VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123.

[1062] In a specific embodiment, the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO:255, HCDR2 of SEQ ID NO:256, HCDR3 of SEQ ID NO:257, LCDR1 of SEQ ID NO:258, LCDR2 of SEQ ID NO:259, and LCDR3 of SEQ ID NO:261.

[1063] In a specific embodiment, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[1064] In a specific embodiment, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively, and the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[1065] In a specific embodiment, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second antigen binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1066] In a specific embodiment, the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively, and the second antigen binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1067] In a specific embodiment, the first antigen binding domain that binds hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[1068] In a specific embodiment, the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the second antigen binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1069] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein

[1070] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 275, 258, 259, and 261, respectively;

[1071] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second binding domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1072] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1073] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1074] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1075] wherein the first domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1076] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1077] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1078] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1079] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1080] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively, and the second domain that binds lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259 and 261, respectively; or

[1081] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, and the second domain that binds lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259 and 261, respectively.

[1082] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1083] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 275, 258, 259, and 261, respectively.

[1084] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1085] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively.

[1086] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1087] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1088] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1089] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively.

[1090] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1091] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1092] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1093] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1094] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1095] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1096] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1097] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1098] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1099] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively.

[1100] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1101] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively.

[1102] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1103] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1104] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1105] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1106] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1107] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein

[1108] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 140, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251;

[1109] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 140, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1110] wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 137 and the VL of SEQ ID NO: 138, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251;

[1111] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 137 and the VL of SEQ ID NO: 138, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1112] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 168 and the VL of SEQ ID NO: 169, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251;

[1113] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251;

[1114] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 166 and the VL of SEQ ID NO: 444, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251;

[1115] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 164 and the VL of SEQ ID NO: 165, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251;

[1116] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 168 and the VL of SEQ ID NO: 169, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1117] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 204 and the VL of SEQ ID NO: 205, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1118] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 204 and the VL of SEQ ID NO: 205, and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251; or

[1119] The first binding domain that binds hK2 comprises VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, and the second binding domain that binds lymphocyte antigen comprises VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251.

[1120] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1121] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 140, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1122] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1123] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 139 and the VL of SEQ ID NO: 140, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1124] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1125] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 137 and the VL of SEQ ID NO: 138, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1126] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1127] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 137 and the VL of SEQ ID NO: 138, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1128] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1129] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 168 and the VL of SEQ ID NO: 169, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1130] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1131] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1132] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1133] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 166 and the VL of SEQ ID NO: 444, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1134] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1135] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 164 and the VL of SEQ ID NO: 165, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1136] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1137] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 168 and the VL of SEQ ID NO: 169, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1138] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1139] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 204 and the VL of SEQ ID NO: 205, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123;

[1140] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1141] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 204 and the VL of SEQ ID NO: 205, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1142] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1143] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds a lymphocyte antigen, wherein the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the second binding domain that binds the lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1144] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein. In some embodiments, the first antigen-binding domain that binds hK2 is conjugated to a first immunoglobulin (Ig) constant region or a fragment of a first Ig constant region, and / or the second antigen-binding domain that binds a lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of a second Ig constant region.

[1145] In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises an Fc region.

[1146] In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region includes a CH2 domain.

[1147] In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region includes a CH3 domain.

[1148] In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises a CH2 domain and a CH3 domain.

[1149] In some embodiments, the fragment of the first Ig constant region and / or the fragment of the second Ig constant region comprises at least a portion of the hinge, a CH2 domain, and a CH3 domain.

[1150] In some embodiments, the fragment of the Ig constant region comprises a hinge, a CH2 domain, and a CH3 domain.

[1151] In some embodiments, the multispecific protein further comprises a second linker (L2) between the first antigen binding domain that binds hK2 and a first Ig constant region or a fragment of a first Ig constant region and the second antigen binding domain that binds a lymphocyte antigen and a second Ig constant region or a fragment of a second Ig constant region.

[1152] In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NO: 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

[1153] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region are of the IgG1, IgG2, and IgG3 or IgG4 isotype.

[1154] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region are of the IgG1 isotype.

[1155] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region are of the IgG2 isotype.

[1156] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region are of the IgG3 isotype.

[1157] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region are of the IgG4 isotype.

[1158] In a specific embodiment, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region are of the IgG1 isotype.

[1159] The first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region can be further engineered as described herein.

[1160] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise at least one mutation that results in reduced binding of the multispecific protein to an FcγR.

[1161] In some embodiments, the at least one mutation that results in reduced binding of the multispecific protein to an FcγR is selected from the group consisting of: F234A / L235A, L234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G23 6 to deletion / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236 to deletion / G237A / P238S, where residues are numbered according to the EU index. In a specific embodiment, the first Ig constant region or a fragment of the first Ig constant region and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations: L234A_L235A_D265S.

[1162] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise at least one mutation that results in enhanced binding of the multispecific protein to an Fcγ receptor (FcγR).

[1163] In some embodiments, the at least one mutation that results in enhanced binding of the multispecific protein to an FcγR is selected from the group consisting of S239D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I332E, where the residues are numbered according to the EU index.

[1164] In some embodiments, the FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRIII, or any combination thereof.

[1165] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise at least one mutation that modulates the half-life of the multispecific protein.

[1166] In some embodiments, the at least one mutation that modulates the half-life of the multispecific protein is selected from the group consisting of: H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H435R, wherein the residues are numbered according to the EU index.

[1167] In some embodiments, the multispecific protein comprises at least one mutation in the CH3 domain of a first Ig constant region or in the CH3 domain of a fragment of a first Ig constant region, and / or at least one mutation in the CH3 domain of a second Ig constant region or in the CH3 domain of a fragment of a second Ig constant region.

[1168] In some embodiments, the at least one mutation in the CH3 domain of the first Ig constant region or in the CH3 domain of a fragment of the first Ig constant region and / or the at least one mutation in the CH3 domain of the second Ig constant region or in the CH3 domain of a fragment of the second Ig constant region is selected from the group consisting of: T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L35 1Y / F405A / Y407V, T366I / K392M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T366L / K392L / T394W, where the residues are numbered according to the EU index. In a specific embodiment, the first Ig constant region or a fragment of the first Ig constant region comprises the following mutations T350V_T366L K392L_T394W and / or the second Ig constant region or a fragment of the second Ig constant region comprises the following mutations T350V_L351Y_F405A_Y407V.

[1169] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations:

[1170] L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig constant region and L235A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig constant region; or

[1171] L235A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig constant region and L235A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig constant region;

[1172] In some embodiments, the first Ig constant region or a fragment of the first Ig constant region and the second Ig constant region or a fragment of the second Ig constant region comprise the following mutations:

[1173] L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the first Ig constant region and L234A_L235A_D265S_T350V_T366L_K392L_T394W in the second Ig constant region; or

[1174] L234A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig constant region and L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig constant region;

[1175] In some embodiments, the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO:378.

[1176] In a specific embodiment, the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises an amino acid sequence identical to SEQ ID NO:378.

[1177] In some embodiments, the first Ig light chain constant region or a fragment of the first Ig light chain constant region comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO:309.

[1178] In a specific embodiment, the first Ig light chain constant region or a fragment of the first Ig light chain constant region comprises an amino acid sequence identical to SEQ ID NO:309.

[1179] In some embodiments, the second Ig constant region or a fragment of the second Ig constant region comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to SEQ ID NO:109.

[1180] In a specific embodiment, the second Ig constant region or fragment of the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO:109.

[1181] In a specific embodiment, the first Ig heavy chain constant region comprises an amino acid sequence identical to SEQ ID NO:378, the first Ig light chain constant region comprises an amino acid sequence identical to SEQ ID NO:309, and the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO:109.

[1182] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378.

[1183] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and a first Ig light chain constant region or a fragment of a first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309.

[1184] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 309.

[1185] In a specific embodiment, the isolated protein disclosed herein comprises a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3) and a second Ig constant region or a fragment of a second Ig constant region comprising an amino acid sequence identical to SEQ ID NO: 109.

[1186] In a specific embodiment, the isolated protein disclosed herein comprises a first antigen binding domain that binds hK2 and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, comprising an amino acid sequence identical to SEQ ID NO: 309, and wherein the isolated protein further comprises a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), and a second Ig constant region, or a fragment of a second Ig constant region, comprising an amino acid sequence identical to SEQ ID NO: 109.

[1187] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region, wherein the first Ig heavy chain constant region comprises an amino acid sequence identical to SEQ ID NO: 378.

[1188] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and a first Ig light chain constant region or a fragment of a first Ig light chain constant region, wherein the first Ig light chain constant region comprises an amino acid sequence identical to SEQ ID NO: 309.

[1189] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, said first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, said first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309.

[1190] In a specific embodiment, the isolated protein disclosed herein comprises: a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261; and a second Ig constant region or a fragment of a second Ig constant region, wherein the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO: 109.

[1191] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively; and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, said first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, said first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309, and wherein the isolated protein further comprises: a second antigen binding domain that binds a lymphocyte antigen, such as CD3, wherein the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261; and a second Ig constant region or a fragment of a second Ig constant region, wherein the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO: 109.

[1192] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region, wherein the first Ig heavy chain constant region comprises an amino acid sequence identical to SEQ ID NO: 378.

[1193] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and a first Ig light chain constant region or a fragment of a first Ig light chain constant region, wherein the first Ig light chain constant region comprises an amino acid sequence identical to SEQ ID NO: 309.

[1194] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and (i) a first Ig heavy chain constant region or a fragment of a first Ig heavy chain constant region, said first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region or a fragment of a first Ig light chain constant region, said first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309.

[1195] In a specific embodiment, the isolated protein disclosed herein comprises: a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the second antigen binding domain that binds a lymphocyte antigen comprises the amino acid sequence of SEQ ID NO: 331; and a second Ig constant region or a fragment of a second Ig constant region, wherein the second Ig constant region comprises an amino acid sequence identical to SEQ ID NO: 109.

[1196] In a specific embodiment, the isolated protein disclosed herein comprises: a first antigen binding domain that binds hK2, wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163; and (i) a first Ig heavy chain constant region, or a fragment of a first Ig heavy chain constant region, the first Ig heavy chain constant region comprising an amino acid sequence identical to SEQ ID NO: 378; and (ii) a first Ig light chain constant region, or a fragment of a first Ig light chain constant region, the first Ig light chain constant region comprising an amino acid sequence identical to SEQ ID NO: 309, and wherein the isolated protein further comprises a second antigen binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the second antigen binding domain that binds a lymphocyte antigen comprises the amino acid sequence of SEQ ID NO: 331 and a second Ig constant region, or a fragment of a second Ig constant region, the second Ig constant region comprising an amino acid sequence identical to SEQ ID NO: 109.

[1197] Generation of multispecific proteins comprising antigen-binding fragments that bind hK2

[1198] The hK2-binding antigen-binding fragments of the present disclosure can be engineered into multispecific antibodies, which are also encompassed within the scope of the present invention.

[1199] Antigen-binding fragments that bind hK2 can be engineered into full-length multispecific antibodies generated using Fab arm exchange, wherein substitutions are introduced into the CH3 domains of the Ig constant regions of two monospecific bivalent antibodies to promote in vitro exchange of Fab arms. In this method, two monospecific bivalent antibodies are engineered to have certain substitutions in the CH3 domains that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to cause disulfide isomerization of cysteines in the hinge region; thereby generating a bispecific antibody through Fab arm exchange. The incubation conditions are ideally returned to non-reducing conditions. Exemplary reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, the following conditions can be used: incubation in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at a pH of 5-8, such as pH 7.0 or pH 7.4, at a temperature of at least 20°C, for at least 90 minutes.

[1200] CH3 mutations that can be used include knob-hole mutations (Genentech), electrostatic matching mutations (Chugai, Amgen, NovoNordisk, Oncomed), strand exchange engineered domain body (SEEDbody) (EMD Serono), Mutagenesis (Genmab) and other asymmetric mutagenesis technologies (such as Zymeworks).

[1201] Knob-hole mutations are disclosed, for example, in WO1996 / 027011 and include mutations at the CH3 interface, where amino acids with small side chains (holes) are introduced into the first CH3 domain, while amino acids with large side chains (knobs) are introduced into the second CH3 domain, resulting in preferential interactions between the first and second CH3 domains. Exemplary CH3 domain mutations that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[1202] The formation of heavy chain heterodimers can be promoted by using electrostatic interactions by replacing positively charged residues on the first CH3 region with negatively charged residues on the second CH3 region, as described in US2010 / 0015133, US2009 / 0182127, US2010 / 028637 or US2011 / 0123532.

[1203] Other asymmetric mutations that can be used to promote heavy chain heterodimerization are L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W, as described in US2012 / 0149876 or US2013 / 0195849 (Zymeworks).

[1204] SEEDbody mutations involve substitution of selected IgG residues for IgA residues to promote heavy chain heterodimerization as described in US20070287170.

[1205] Other exemplary mutations that can be used are R409D_K370E / D399K_E357K, S354C_T366W / Y349C_T366S_L368A_Y407V, Y349C_T366W / S354C_T366S_L368A_Y407V, T366K / L351D, L351K / Y349E, L351K / Y34 9D, L351K / L368E, L351Y__Y407A / T366A_K409F, L351Y__Y407A / T366V_K409F, K392D / D399K, K392D / E356K, K253E__D282K_K322D / D239K_E240K_K292D, K392DK409D / D356K D399K, as described in WO2007 / 147901, WO2011 / 143545, WO2013157954, WO2013096291 and US2018 / 0118849.

[1206] Mutations (Genmab) are disclosed in, for example, US9150663 and US2014 / 0303356, and include mutations F405L / K409R, wild type / F405L_R409K, T350I_K370T_F405L / K409R, K370W / K409R, D399AFGHILMNRSTVWY / K409R, T366ADEFGHILMQVY / K409R, L368ADEGHNRSTVQ / K409AGRH, D399FHKRQ / K409AGRH, F405IKLSTVW / K409AGRH, and Y407LWQ / K409AGRH.

[1207] Additional bispecific or multispecific structures into which an antigen binding domain that binds hK2 can be incorporated include dual variable domain immunoglobulins (DVDs) (International Patent Publication No. WO2009 / 134776; DVDs are full-length antibodies comprising a heavy chain having a VH1-linker-VH2-CH structure and a light chain having a VL1-linker-VL2-CL structure; the linker is optional), structures comprising a variety of dimerization domains to connect two antibody arms with different specificities, such as leucine zippers or collagen dimerization domains (International Patent Publication No. WO2012 / 022811, U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441), two or more domain antibodies (dAbs) conjugated together, bivalent antibodies, heavy chain-only antibodies such as camelid antibodies and engineered camelid antibodies, dual targeting (DT)-Igs (GSK / Domantis), two-in-one antibodies (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-host (CovX / Pfizer), IgG-like bispecific antibodies (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche), ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), dual affinity retargeting technology (Fc-DART) (MacroGenics) and bis(ScFv)2-Fab (National Research Center for Antibody Medicine--China), bifunctional or Bis-Fab (Genentech), dock-lock (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech). ScFv-based, diabody-based domain antibodies include, but are not limited to, bispecific T-cell engager (BiTE) (Micromet), tandem diabody (Tandab) (Affimed), dual affinity retargeting technology (DART) (MacroGenics), single-chain diabody (Academic), TCR-like antibody (AIT, ReceptorLogics), human serum albumin ScFv fusion (Merrimack) and COMBODY (Epigen Biotech), dual-targeting nanobody (Ablynx), dual-targeting heavy chain domain-only antibody.

[1208] The hK2-binding antigen-binding domains disclosed herein can also be engineered into multispecific proteins comprising three polypeptide chains. In such designs, at least one antigen-binding domain is in the form of an scFv. Exemplary designs include (where "1" indicates the first antigen-binding domain, "2" indicates the second antigen-binding domain, and "3" indicates the third antigen-binding domain):

[1209] Design 1: Chain A) scFv1-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3

[1210] Design 2: Chain A) scFv1-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3

[1211] Design 3: Chain A) scFv1-CH1-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3

[1212] Design 4: Chain A) CH2-CH3-scFv1; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3

[1213] CH3 can be engineered into designs 1-4, such as mutations L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A K409F or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W as described in US2012 / 0149876 or US2013 / 0195849 (Zymeworks).

[1214] In a specific embodiment, the design is: Chain A) scFv-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3.

[1215] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding domain that binds hK2 comprises a HCDR1 of SEQ ID NO: 63, 72, 141, 147, 170, 176, 188, 194, 196, 198, 200, 206, or 216, a HCDR2 of SEQ ID NO: 64, 65, 73, 142, 148, 171, 177, 188, 189, 195, 197, 199, 201, 207, or 217, a HCDR3 of SEQ ID NO: 66, 143, 172, 178, 184, 190, 208, or 218, a LCDR1 of SEQ ID NO: 67, 68, 144, 173, 179, 182, 185, or 191, a LCDR2 of SEQ ID NO: 69, 70, 145, 174, 180, 186, 192 or 470 and LCDR3 of SEQ ID NO: 71, 146, 175, 181, 187, 193 or 209.

[1216] In some embodiments, the lymphocyte antigen is CD3. In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the following sequence:

[1217] SEQ ID NOs: 141, 142, 143, 144, 145 and 146, respectively;

[1218] SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively;

[1219] SEQ ID NOs: 176, 177, 178, 179, 180 and 181, respectively;

[1220] SEQ ID NOs: 170, 183, 184, 185, 186 and 187, respectively;

[1221] SEQ ID NOs: 188, 189, 190, 191, 192 and 193, respectively;

[1222] SEQ ID NOs: 206, 207, 208, 182, 470, and 209, respectively;

[1223] SEQ ID NOs: 147, 148, 143, 144, 145 and 146, respectively;

[1224] SEQ ID NOs: 194, 195, 172, 173, 174 and 175, respectively;

[1225] SEQ ID NOs: 196, 197, 178, 179, 190 and 181, respectively;

[1226] SEQ ID NOs: 198, 199, 184, 185, 186 and 187, respectively;

[1227] SEQ ID NOs: 200, 201, 190, 191, 192, and 193, respectively; or

[1228] They are SEQ ID NOs: 216, 217, 218, 182, 470 and 209, respectively.

[1229] In some embodiments, the lymphocyte antigen is CD3. In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, or SEQ ID NOs: 194, 195, 172, 173, 174, and 175, respectively.

[1230] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding domain that binds hK2 comprises:

[1231] VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138;

[1232] VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163;

[1233] VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165;

[1234] VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167;

[1235] VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169; or

[1236] The VH of SEQ ID NO: 204 and the VL of SEQ ID NO: 205.

[1237] In some embodiments, the lymphocyte antigen is CD3.

[1238] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 74.

[1239] In some embodiments, the lymphocyte antigen is CD3. In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[1240] In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding domain that binds hK2 comprises a VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to a VL of SEQ ID NO: 163.

[1241] In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163.

[1242] In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding domain that binds hK2 comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 162 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 163.

[1243] In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163.

[1244] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding domain that binds hK2 comprises: a VH of SEQ ID NO: 4, 5, 6, 137, 139, 159, or 161 and a VL of SEQ ID NO: 1, 2, 3, 140, or 160, with the proviso that the antigen binding domain that binds hK2 does not comprise both the VH of SEQ ID NO: 5 and the VL of SEQ ID NO: 2.

[1245] In some embodiments, the lymphocyte antigen is CD3.

[1246] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding domain that binds hK2 comprises:

[1247] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1;

[1248] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2;

[1249] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3;

[1250] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140;

[1251] VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160;

[1252] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1;

[1253] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3;

[1254] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140;

[1255] VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160;

[1256] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1;

[1257] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2;

[1258] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3;

[1259] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140;

[1260] VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160;

[1261] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1;

[1262] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2;

[1263] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3;

[1264] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140;

[1265] VH of SEQ ID NO: 139 and VL of SEQ ID NO: 160;

[1266] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1;

[1267] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2;

[1268] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3;

[1269] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140;

[1270] VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160;

[1271] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1;

[1272] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2;

[1273] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3;

[1274] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or

[1275] VH of SEQ ID NO: 161 and VL of SEQ ID NO: 160.

[1276] In some embodiments, the lymphocyte antigen is CD3.

[1277] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 133, 134, 135, 136, 308, 316, 318, 319, 320, 321, 322, 323, 324, 325, 404, 405, 406, 407, 408, or 409.

[1278] In some embodiments, the lymphocyte antigen is CD3.

[1279] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen-binding domain that binds hK2 comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID SEQ: 404 or 405.

[1280] In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen (eg, CD3), wherein the first antigen binding domain that binds hK2 comprises the amino acid sequence of SEQ ID NO: 404 or 405.

[1281] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises a HCDR1 of SEQ ID NO: 255 or 116, a HCDR2 of SEQ ID NO: 256 or 117, a HCDR3 of SEQ ID NO: 257 or 118, a LCDR1 of SEQ ID NO: 258 or 119, a LCDR2 of SEQ ID NO: 259 or 120, and a LCDR3 of SEQ ID NO: 260, 261 or 121.

[1282] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 157.

[1283] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises:

[1284] HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 260; or

[1285] HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[1286] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises:

[1287] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 249;

[1288] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 250;

[1289] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 251;

[1290] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 252;

[1291] VH of SEQ ID NO: 248 and VL of SEQ ID NO: 253; or

[1292] The VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 254.

[1293] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises a VH that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VH of SEQ ID NO: 248 and a VL that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the VL of SEQ ID NO: 251.

[1294] In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251.

[1295] In some embodiments, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises:

[1296] HCDR1 of SEQ ID NO: 116, HCDR2 of SEQ ID NO: 117, HCDR3 of SEQ ID NO: 118, LCDR1 of SEQ ID NO: 119, LCDR2 of SEQ ID NO: 120, and LCDR3 of SEQ ID NO: 121; or

[1297] VH of SEQ ID NO: 122 and VL of SEQ ID NO: 123.

[1298] In a specific embodiment, the isolated multispecific protein comprises a first binding domain that binds hK2 and a second binding domain that binds a lymphocyte antigen, wherein the second antigen binding domain that binds a lymphocyte antigen comprises HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 261.

[1299] In some embodiments, the second binding domain that binds a lymphocyte antigen of the isolated multispecific protein comprises the amino acid sequence of SEQ ID NO: 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, or 337.

[1300] In a specific embodiment, the second binding domain that binds a lymphocyte antigen of the isolated multispecific protein comprises an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) identical to the amino acid sequence of SEQ ID NO:331.

[1301] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1302] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 275, 258, 259, and 260, respectively;

[1303] the first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 136 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251; and / or

[1304] The isolated multispecific protein comprises HCl of SEQ ID NO:351, HC2 of SEQ ID NO:358, and LC2 of SEQ ID NO:267.

[1305] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1306] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1307] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1308] The first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 136 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123; and / or

[1309] The isolated multispecific protein comprises HCl of SEQ ID NO:351, HC2 of SEQ ID NO:359, and LC2 of SEQ ID NO:272.

[1310] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1311] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1312] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively;

[1313] the first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 134 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251; and / or

[1314] The isolated multispecific protein comprises HCl of SEQ ID NO:352, HC2 of SEQ ID NO:358, and LC2 of SEQ ID NO:267.

[1315] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1316] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1317] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1318] The first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 134 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123; and / or

[1319] The isolated multispecific protein comprises HCl of SEQ ID NO:352, HC2 of SEQ ID NO:259, and LC2 of SEQ ID NO:272.

[1320] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1321] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1322] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively;

[1323] the first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 325 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 251; and / or

[1324] The isolated multispecific protein comprises HCl of SEQ ID NO:353, HC2 of SEQ ID NO:358, and LC2 of SEQ ID NO:267.

[1325] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1326] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1327] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively;

[1328] The first binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163 and the second binding domain that binds a lymphocyte antigen comprises the scFv of SEQ ID NO: 331; and / or

[1329] The isolated multispecific protein comprises HCl of SEQ ID NO:354, LC1 of SEQ ID NO:221, and HC2 of SEQ ID NO:360.

[1330] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1331] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1332] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively;

[1333] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 166 and the VL of SEQ ID NO: 444 and the second binding domain that binds a lymphocyte antigen comprises the scFv of SEQ ID NO: 331; and / or

[1334] The isolated multispecific protein comprises HCl of SEQ ID NO:355, LC1 of SEQ ID NO:445, and HC2 of SEQ ID NO:360.

[1335] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1336] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1337] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively;

[1338] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 164 and the VL of SEQ ID NO: 165 and the second binding domain that binds a lymphocyte antigen comprises the scFv of SEQ ID NO: 331; and / or

[1339] The isolated multispecific protein comprises HCl of SEQ ID NO:356, LC1 of SEQ ID NO:222, and HC2 of SEQ ID NO:360.

[1340] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1341] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1342] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1343] The first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 325 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123; and / or

[1344] The isolated multispecific protein comprises HCl of SEQ ID NO:353, HC2 of SEQ ID NO:359, and LC2 of SEQ ID NO:272.

[1345] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1346] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1347] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1348] The first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 316 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 122 and the VL of SEQ ID NO: 123; and / or

[1349] The isolated multispecific protein comprises HCl of SEQ ID NO:357, HC2 of SEQ ID NO:359, and LC2 of SEQ ID NO:272.

[1350] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1351] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1352] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively;

[1353] the first binding domain that binds hK2 comprises the scFv of SEQ ID NO: 316 and the second binding domain that binds a lymphocyte antigen comprises the VH of SEQ ID NO: 248 and the VL of SEQ ID NO: 151; and / or

[1354] The isolated multispecific protein comprises HCl of SEQ ID NO:357, HC2 of SEQ ID NO:358, and LC2 of SEQ ID NO:267.

[1355] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1356] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1357] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively;

[1358] the first binding domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163 and the second binding domain that binds a lymphocyte antigen comprises the scFv of SEQ ID NO: 331; and / or

[1359] The isolated multispecific protein comprises HCl of SEQ ID NO:361, LC1 of SEQ ID NO:221, and HC2 of SEQ ID NO:362.

[1360] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1361] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein

[1362] the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 275, 258, 259, and 261, respectively;

[1363] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second binding domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1364] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1365] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1366] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1367] wherein the first domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1368] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1369] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively;

[1370] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1371] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, and 209, respectively, and the second domain that binds lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively;

[1372] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 206, 207, 208, 182, 470, 209, respectively, and the second domain that binds lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259 and 261, respectively; or

[1373] wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, and the second domain that binds lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259 and 261, respectively.

[1374] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1375] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 275, 258, 259, and 261, respectively.

[1376] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1377] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 63, 65, 66, 67, 69, and 71, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively.

[1378] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1379] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1380] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1381] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 141, 142, 143, 144, 145, and 146, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 116, 117, 118, 119, 120, and 121, respectively.

[1382] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1383] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 188, 189, 190, 191, 192, and 193, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1384] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1385] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174, and 175, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1386] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1387] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 170, 183, 184, 185, 186, and 187, respectively, and the second domain that binds a lymphocyte antigen comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259, and 261, respectively.

[1388] In some embodiments, the isolated multispecific protein is an anti-hK2 / anti-CD3 protein.

[1389] The present disclosure also provides an isolated multispecific protein comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first binding domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 176, 177, 178, 179, 180, and 181, respectively, and the second domain that binds a lymphocyte antigen compri...

Claims

1. An isolated anti-hK2 / anti-CD3 antibody comprising a first domain that binds hK2 and a second domain that binds CD3, wherein a. the first domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, and the second domain that binds CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259 and 261, respectively; and / or b. the first domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the second domain that binds CD3 comprises the scFv of SEQ ID NO: 331; and / or c. The isolated anti-hK2 / anti-CD3 antibody comprises HC1 of SEQ ID NO: 354, LC1 of SEQ ID NO: 221, and HC2 of SEQ ID NO:

360.

2. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the isolated anti-hK2 / anti-CD3 antibody comprises a lysine at the C-terminus of HC1 and HC2, and wherein the isolated anti-hK2 / anti-CD3 antibody comprises an HC1 that is at least 80% identical to the HC1 of SEQ ID NO: 361 and an HC2 that is at least 80% identical to the HC2 of SEQ ID NO:

362.

3. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the first domain that binds hK2 and / or the second domain that binds CD3 in item a comprises scFv, (scFv)2, Fv, Fab or F(ab')2.

4. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the first domain that binds hK2 in item b comprises scFv, (scFv)2, Fv, Fab or F(ab')2.

5. The isolated anti-hK2 / anti-CD3 antibody of claim 3 or 4, wherein the first domain that binds hK2 comprises a Fab and the second domain that binds CD3 comprises a scFv.

6. The isolated anti-hK2 / anti-CD3 antibody of claim 5, wherein the scFv comprises, from N-terminus to C-terminus, VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH).

7. The isolated anti-hK2 / anti-CD3 antibody of claim 6, wherein the L1 comprises: a.5-50 amino acids; b.5-40 amino acids; c. 10-30 amino acids; or d. 10-20 amino acids.

8. The isolated anti-hK2 / anti-CD3 antibody of claim 7, wherein the L1 comprises the amino acid sequence of SEQ ID NO:7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

9. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the isolated anti-hK2 / anti-CD3 antibody comprises a lysine at the C-terminus of HC1 and HC2, and wherein the isolated anti-hK2 / anti-CD3 antibody comprises an HC1 that is at least 95% identical to the HC1 of SEQ ID NO: 361 and an HC2 that is at least 95% identical to the HC2 of SEQ ID NO:

362.

10. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the isolated anti-hK2 / anti-CD3 antibody comprises a lysine at the C-terminus of HC1 and HC2, and wherein the isolated anti-hK2 / anti-CD3 antibody comprises an HC1 that is at least 99% identical to the HC1 of SEQ ID NO: 361 and an HC2 that is at least 99% identical to the HC2 of SEQ ID NO:

362.

11. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the first domain that binds hK2 comprises a VH at least 95% identical to the VH of SEQ ID NO: 162 and a VL at least 95% identical to the VL of SEQ ID NO: 163, and the second domain that binds CD3 comprises a scFv at least 95% identical to the scFv of SEQ ID NO:

331.

12. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the first domain that binds hK2 comprises a VH that is at least 99% identical to the VH of SEQ ID NO: 162 and a VL that is at least 99% identical to the VL of SEQ ID NO: 163, and the second domain that binds CD3 comprises a scFv that is at least 99% identical to the scFv of SEQ ID NO:

331.

13. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the isolated anti-hK2 / anti-CD3 antibody comprises an HC1 at least 95% identical to the HC1 of SEQ ID NO: 354, an LC1 at least 95% identical to the LC1 of SEQ ID NO: 221, and an HC2 at least 95% identical to the HC2 of SEQ ID NO:

360.

14. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the isolated anti-hK2 / anti-CD3 antibody comprises an HC1 at least 99% identical to the HC1 of SEQ ID NO:354, an LC1 at least 99% identical to the LC1 of SEQ ID NO:221, and an HC2 at least 99% identical to the HC2 of SEQ ID NO:

360.

15. The isolated anti-hK2 / anti-CD3 antibody of claim 1, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) heavy chain constant region or a fragment of the first Ig heavy chain constant region, and The second domain that binds CD3 is conjugated to a second immunoglobulin (Ig) heavy chain constant region or a fragment of the second Ig heavy chain constant region.

16. The isolated anti-hK2 / anti-CD3 antibody of claim 15, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region are of the IgGl isotype.

17. The isolated anti-hK2 / anti-CD3 antibody of claim 16, wherein the antibody comprises the following amino acid mutations: L234A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region, and L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig heavy chain constant region or the fragment of the second Ig heavy chain constant region, The numbering of the amino acid mutations is according to the EU index.

18. The isolated anti-hK2 / anti-CD3 antibody of claim 15, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

378.

19. The isolated anti-hK2 / anti-CD3 antibody of claim 15, wherein the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

109.

20. The isolated anti-hK2 / anti-CD3 antibody of claim 15, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 378, and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

109.

21. The isolated anti-hK2 / anti-CD3 antibody of claim 15, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:378, and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

109.

22. The isolated anti-hK2 / anti-CD3 antibody of claim 15, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:378, and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

109.

23. The isolated anti-hK2 / anti-CD3 antibody of claim 20, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region, and The first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region comprises the amino acid sequence of SEQ ID NO:

309.

24. The isolated anti-hK2 / anti-CD3 antibody of claim 21, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region, and The first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

309.

25. The isolated anti-hK2 / anti-CD3 antibody of claim 22, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region, and The first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

309.

26. A pharmaceutical composition comprising the isolated anti-hK2 / anti-CD3 antibody of claim 1 and a pharmaceutically acceptable carrier.

27. Use of the isolated anti-hK2 / anti-CD3 antibody of claim 1 in the preparation of a medicament for treating an hK2-expressing cancer in a subject.

28. The use of claim 27, wherein the cancer expressing hK2 is prostate cancer.

29. The use of claim 27, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

30. The use of claim 28, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

31. The use of claim 28, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

32. The use of claim 31, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

33. An isolated anti-hK2 / anti-CD3 antibody comprising a first domain that binds hK2 and a second domain that binds CD3, wherein a. the first domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, and the second domain that binds CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259 and 261, respectively; and / or b. the first domain that binds hK2 comprises the VH of SEQ ID NO: 162 and the VL of SEQ ID NO: 163, and the second domain that binds CD3 comprises the scFv of SEQ ID NO: 331; and / or c. The isolated anti-hK2 / anti-CD3 antibody comprises HC1 of SEQ ID NO:361, LC1 of SEQ ID NO:221, and HC2 of SEQ ID NO:

362.

34. The isolated anti-hK2 / anti-CD3 antibody of claim 33, wherein the first domain that binds hK2 and / or the second domain that binds CD3 in item a comprises scFv, (scFv)2, Fv, Fab or F(ab')2.

35. The isolated anti-hK2 / anti-CD3 antibody of claim 33, wherein the first domain that binds hK2 in item b comprises scFv, (scFv)2, Fv, Fab, or F(ab')2.

36. The isolated anti-hK2 / anti-CD3 antibody of claim 34 or 35, wherein the first domain that binds hK2 comprises a Fab and the second domain that binds CD3 comprises a scFv.

37. The isolated anti-hK2 / anti-CD3 antibody of claim 36, wherein the scFv comprises, from N-terminus to C-terminus, VH, a first linker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L1-VH).

38. The isolated anti-hK2 / anti-CD3 antibody of claim 37, wherein the L1 comprises: a.5-50 amino acids; b.5-40 amino acids; c. 10-30 amino acids; or d. 10-20 amino acids.

39. The isolated anti-hK2 / anti-CD3 antibody of claim 38, wherein the L1 comprises the amino acid sequence of SEQ ID NO:7, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

40. The isolated anti-hK2 / anti-CD3 antibody of claim 33, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) heavy chain constant region or a fragment of the first Ig heavy chain constant region, and The second domain that binds CD3 is conjugated to a second immunoglobulin (Ig) heavy chain constant region or a fragment of the second Ig heavy chain constant region.

41. The isolated anti-hK2 / anti-CD3 antibody of claim 40, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region are of the IgGl isotype.

42. The isolated anti-hK2 / anti-CD3 antibody of claim 41, wherein the antibody comprises the following amino acid mutations: L234A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region, and L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig heavy chain constant region or the fragment of the second Ig heavy chain constant region, The numbering of the amino acid mutations is according to the EU index.

43. The isolated anti-hK2 / anti-CD3 antibody of claim 40, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

378.

44. The isolated anti-hK2 / anti-CD3 antibody of claim 40, wherein the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

109.

45. The isolated anti-hK2 / anti-CD3 antibody of claim 40, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 378, and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

109.

46. ​​The isolated anti-hK2 / anti-CD3 antibody of claim 40, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:378, and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

109.

47. The isolated anti-hK2 / anti-CD3 antibody of claim 40, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:378, and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

109.

48. The isolated anti-hK2 / anti-CD3 antibody of claim 45, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region, and The first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region comprises the amino acid sequence of SEQ ID NO:

309.

49. The isolated anti-hK2 / anti-CD3 antibody of claim 46, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region, and The first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

309.

50. The isolated anti-hK2 / anti-CD3 antibody of claim 47, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region, and The first immunoglobulin (Ig) light chain constant region or a fragment of the first Ig light chain constant region comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:

309.

51. A pharmaceutical composition comprising the isolated anti-hK2 / anti-CD3 antibody of claim 33 and a pharmaceutically acceptable carrier.

52. Use of the isolated anti-hK2 / anti-CD3 antibody of claim 33 in the preparation of a medicament for treating an hK2-expressing cancer in a subject.

53. The use of claim 52, wherein the cancer expressing hK2 is prostate cancer.

54. The use of claim 52, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

55. The use of claim 53, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

56. The use of claim 53, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

57. The use of claim 56, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

58. An isolated anti-hK2 / anti-CD3 antibody, comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first domain that binds hK2 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 170, 171, 172, 173, 174 and 175, respectively, and the second domain that binds CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NOs: 255, 256, 257, 258, 259 and 261, respectively.

59. The isolated anti-hK2 / anti-CD3 antibody of claim 58, wherein the first domain that binds hK2 comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163, and the second domain that binds CD3 comprises a scFv of SEQ ID NO:

331.

60. The isolated anti-hK2 / anti-CD3 antibody of claim 58, wherein the isolated anti-hK2 / anti-CD3 antibody comprises HC1 of SEQ ID NO: 354, LC1 of SEQ ID NO: 221, and HC2 of SEQ ID NO:

360.

61. The isolated anti-hK2 / anti-CD3 antibody of claim 58, wherein the isolated anti-hK2 / anti-CD3 antibody comprises HC1 of SEQ ID NO:361, LC1 of SEQ ID NO:221, and HC2 of SEQ ID NO:

362.

62. The isolated anti-hK2 / anti-CD3 antibody of claim 58, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) heavy chain constant region or a fragment of the first Ig heavy chain constant region, and The second domain that binds CD3 is conjugated to a second immunoglobulin (Ig) heavy chain constant region or a fragment of the second Ig heavy chain constant region.

63. The isolated anti-hK2 / anti-CD3 antibody of claim 62, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region are of the IgGl isotype.

64. The isolated anti-hK2 / anti-CD3 antibody of claim 63, wherein the antibody comprises the following amino acid mutations: L234A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region, and L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region, The numbering of the amino acid mutations is according to the EU index.

65. A pharmaceutical composition comprising the isolated anti-hK2 / anti-CD3 antibody of claim 58 and a pharmaceutically acceptable carrier.

66. Use of the isolated anti-hK2 / anti-CD3 antibody of claim 58 in the preparation of a medicament for treating an hK2-expressing cancer in a subject.

67. The use of claim 66, wherein the hK2-expressing cancer is prostate cancer.

68. The use of claim 66, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

69. The use of claim 67, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

70. The use of claim 67, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

71. The use of claim 70, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

72. An isolated anti-hK2 / anti-CD3 antibody, comprising a first domain that binds hK2 and a second domain that binds CD3, wherein the first domain that binds hK2 comprises a VH of SEQ ID NO: 162 and a VL of SEQ ID NO: 163, and the second domain that binds CD3 comprises a scFv of SEQ ID NO:

331.

73. The isolated anti-hK2 / anti-CD3 antibody of claim 72, wherein the isolated anti-hK2 / anti-CD3 antibody comprises HC1 of SEQ ID NO: 354, LC1 of SEQ ID NO: 221, and HC2 of SEQ ID NO:

360.

74. The isolated anti-hK2 / anti-CD3 antibody of claim 72, wherein the isolated anti-hK2 / anti-CD3 antibody comprises HC1 of SEQ ID NO:361, LC1 of SEQ ID NO:221, and HC2 of SEQ ID NO:

362.

75. The isolated anti-hK2 / anti-CD3 antibody of claim 72, wherein the first domain that binds hK2 is conjugated to a first immunoglobulin (Ig) heavy chain constant region or a fragment of the first Ig heavy chain constant region, and The second domain that binds CD3 is conjugated to a second immunoglobulin (Ig) heavy chain constant region or a fragment of the second Ig heavy chain constant region.

76. The isolated anti-hK2 / anti-CD3 antibody of claim 75, wherein the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region and the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region are of the IgGl isotype.

77. The isolated anti-hK2 / anti-CD3 antibody of claim 76, wherein the antibody comprises the following amino acid mutations: L234A_L235A_D265S_T350V_T366L_K392L_T394W in the first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region, and L234A_L235A_D265S_T350V_L351Y_F405A_Y407V in the second Ig heavy chain constant region or a fragment of the second Ig heavy chain constant region, The numbering of the amino acid mutations is according to the EU index.

78. A pharmaceutical composition comprising the isolated anti-hK2 / anti-CD3 antibody of claim 72 and a pharmaceutically acceptable carrier.

79. Use of the isolated anti-hK2 / anti-CD3 antibody of claim 72 in the preparation of a medicament for treating an hK2-expressing cancer in a subject.

80. The use of claim 79, wherein the hK2-expressing cancer is prostate cancer.

81. The use of claim 79, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

82. The use of claim 80, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

83. The use of claim 80, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

84. The use of claim 83, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

85. An isolated anti-hK2 / anti-CD3 antibody comprising HC1 of SEQ ID NO:354, LC1 of SEQ ID NO:221, and HC2 of SEQ ID NO:

360.

86. A pharmaceutical composition comprising the isolated anti-hK2 / anti-CD3 antibody of claim 85 and a pharmaceutically acceptable carrier.

87. Use of the isolated anti-hK2 / anti-CD3 antibody of claim 85 in the preparation of a medicament for treating an hK2-expressing cancer in a subject.

88. The use of claim 87, wherein the cancer expressing hK2 is prostate cancer.

89. The use of claim 86, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

90. The use of claim 88, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

91. The use of claim 88, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

92. The use of claim 91, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

93. An isolated anti-hK2 / anti-CD3 antibody comprising HC1 of SEQ ID NO:361, LC1 of SEQ ID NO:221, and HC2 of SEQ ID NO:

362.

94. A pharmaceutical composition comprising the isolated anti-hK2 / anti-CD3 antibody of claim 93 and a pharmaceutically acceptable carrier.

95. Use of the isolated anti-hK2 / anti-CD3 antibody of claim 93 in the preparation of a medicament for treating an hK2-expressing cancer in a subject.

96. The use of claim 95, wherein the cancer expressing hK2 is prostate cancer.

97. The use of claim 95, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

98. The use of claim 96, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

99. The use of claim 96, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

100. The use of claim 99, wherein the antibody is administered in combination with at least one additional therapeutic agent, and wherein the at least one additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy, or radiation therapy, or any combination thereof.

101. A polynucleotide encoding the isolated anti-hK2 / anti-CD3 antibody of any one of claims 1-25, 33-50, 58-64, 72-77, 85 or 93.

102. A vector comprising the polynucleotide of claim 101.

103. A host cell comprising the vector of claim 102.

104. A method of producing the isolated anti-hK2 / anti-CD3 antibody of any one of claims 1-25, 33-50, 58-64, 72-77, 85 or 93, the method comprising culturing the host cell of claim 103 under conditions such that the isolated anti-hK2 / anti-CD3 antibody is expressed, and recovering the isolated anti-hK2 / anti-CD3 antibody produced by the host cell.

105. Use of the pharmaceutical composition of any one of claims 26, 51, 65, 78, 86 or 94 in the preparation of a medicament for treating an hK2-expressing cancer in a subject.

106. The use of claim 105, wherein the cancer expressing hK2 is prostate cancer.

107. The use of claim 106, wherein the prostate cancer is recurrent, refractory, malignant or castration-resistant prostate cancer, or any combination thereof.

108. The use of claim 105, wherein the pharmaceutical composition is administered in combination with at least one additional therapeutic agent.

109. The use of claim 108, wherein the additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy or radiation therapy, or any combination thereof.

110. The use of claim 106, wherein the pharmaceutical composition is administered in combination with at least one additional therapeutic agent.

111. The use of claim 110, wherein the additional therapeutic agent is surgery, chemotherapy, androgen deprivation therapy or radiation therapy, or any combination thereof.

112. A kit comprising the isolated anti-hK2 / anti-CD3 antibody of any one of claims 1-25, 33-50, 58-64, 72-77, 85 or 93, the immunoconjugate of claim 99, or the pharmaceutical composition of any one of claims 26, 51, 65, 78, 86 or 94.

Images