Anti-PTK7 antibodies, conjugates and methods of use

Abstract

Antibody constructs that bind to human PTK7, including monospecific and bispecific anti-PTK7 antibody constructs. Bispecific anti-PTK7 antibody constructs may bind to PTK7 and another non- PTK7 protein, or they may bind to two different epitopes on PTK7 (biparatopic anti-PTK7 antibody constructs). Antibody-drug conjugates (ADCs) comprising the anti-PTK7 antibody constructs conjugated to one or more drug moieties are also described. The anti-PTK7 antibody constructs and ADCs may find use as therapeutics or diagnostics.

Description

ANTI-PTK7 ANTIBODIES, CONJUGATES AND METHODS OF USEFIELD

[0001] The present disclosure relates to the field of antibody therapeutics and, in particular, to antibodies that bind to PTK7, including biparatopic anti-PTK7 antibodies, conjugates comprising the antibodies and methods of using the antibodies and conjugates.BACKGROUND

[0002] Protein Tyrosine Kinase 7 (PTK7), also known as colon carcinoma kinase 4 (CCK4), is an evolutionarily conserved transmembrane receptor involved in tissue development and homeostasis. PTK7 is upregulated in various types of cancer, such as breast cancer, colorectal cancer, lung cancers, gastric cancer, esophageal cancer and cholangiocarcinoma.

[0003] Anti-PTK7 antibodies and antibody-drug conjugates have been described (U. S. Patent No. 8,222,395; International Patent Application Nos: WO 2012 / 112943 and WO 2015 / 168019). The anli-PI’K7 ADC, cofetuzumab pclidolin, is in development for the treatment of cancer and comprises an anti-PTK7 antibody conjugated to an auristatin payload (see Maitland, et al., 2021, Clin Cancer Res, 27(16):4511-4520).

[0004] Camptothecin analogues have been developed as payloads for ADCs. For example, trastuzumab deruxtecan (Enhertu™) in which the camptothecin analogue, deruxtecan (Dxd), is conjugated to the anti-HER2 antibody, trastuzumab, via a cleavable tetrapeptide-based linker, and sacituzumab govitecan (Trodelvy™) in which the camptothecin analogue, SN-38, is conjugated to the anti-Trop-2 antibody, sacituzumab, via a hydrolysable, pH-sensitive linker, have both been approved for treatment of cancer.

[0005] Other camptothecin analogues and derivatives, as well as ADCs comprising them have been described. See, for example, International (PCT) Application Publication Nos. WO 2019 / 195665, WO 2019 / 236954, WO 2020 / 200880, WO 2020 / 219287 and WO 2022 / 246576.

[0006] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission isnecessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the claimed invention.SUMMARY

[0007] Described herein are anti-PTK7 antibodies, conjugates comprising the anti-PTK7 antibodies and methods of using the anti-PTK7 antibodies and conjugates. One aspect of the present disclosure relates to an antibody construct comprising a first antigen-binding domain that binds to human PTK7 and comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36, and a second antigen-binding domain that binds to human PTK7 and comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64, wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0008] Another aspect of the present disclosure relates to an antibody construct comprising: a first antigen-binding domain that binds to human PTK7 and comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 73, and a light chain comprising the sequence as set forth in SEQ ID NO: 75, and a second antigen-binding domain that binds to human PTK7 and comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 74, and a light chain comprising the sequence as set forth in SEQ ID NO: 76.

[0009] Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that binds to human PTK7, wherein the antigen-binding domain comprises the CDR (HCDR1, HCDR2, HCDR3) sequences of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the CDR (LCDR1, LCDR2, LCDR3) sequences of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36, and wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0010] Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that binds to human PTK7, wherein the antigen-binding domain comprises the CDR (HCDR1, HCDR2, HCDR3) sequences of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the CDR (LCDR1, LCDR2, LCDR3) sequences of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64, and wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0011] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding an antibody construct as described herein.

[0012] Another aspect of the present disclosure relates to an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding an antibody construct as described herein.

[0013] Another aspect of the present disclosure relates to host cell comprising a polynucleotide or set of polynucleotides encoding an antibody construct as described herein or an expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides.

[0014] Another aspect of the present disclosure relates to an antibody-drug conjugate comprising an antibody construct as described herein conjugated to one or more drug moieties.

[0015] Another aspect of the present disclosure relates to an antibody-drug conjugate having general Formula (I): A-(L-(D)m)n (I), wherein: A is an antibody construct as described herein; L is a linker; D is a drug moiety; m is between 1 and about 8, and n is 1 and about 12.

[0016] Another aspect of the present disclosure relates to a method of preparing an antibodydrug conjugate having general Formula (I): A-(L-(D)m)n (I), comprising conjugating drug-linker L-(D)mto the antibody construct, A.

[0017] Another aspect of the present disclosure relates to an antibody-drug conjugate having the structure:wherein n is between about 4 and about 8, and wherein A is an antibody construct comprising: (a) the first antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 10; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 11; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 12; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 23; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 24, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 22; and (b) the second antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 40; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 92; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 42; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 53; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 54, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 52.Another aspect of the present disclosure relates to a method of preparing an antibody-drug conjugate having the structure:comprising conjugating a drug-linker having the structure:to the antibody construct, A, wherein the drug-linker is conjugated to native cysteine residues of the antibody construct, A.

[0018] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody construct as described herein, and a pharmaceutically acceptable carrier or diluent.

[0019] Another aspect of the present disclosure relates to pharmaceutical composition comprising an antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier or diluent.

[0020] Another aspect of the present disclosure relates to an antibody construct as described herein for use in therapy.

[0021] Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in therapy.

[0022] Another aspect of the present disclosure relates to a use of an antibody construct as described herein in the manufacture of a medicament for the treatment of cancer.

[0023] Another aspect of the present disclosure relates to a use of an antibody-drug conjugate as described herein in the manufacture of a medicament for the treatment of cancer.

[0024] Another aspect of the present disclosure relates to a method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody construct as described herein.

[0025] Another aspect of the present disclosure relates to a method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody-drug conjugate as described herein.

[0026] Another aspect of the present disclosure relates to a method of treating a subject having a cancer comprising administering to the subject an effective amount of an antibody construct as described herein.

[0027] Another aspect of the present disclosure relates to a method of treating a subject having a cancer comprising administering to the subject an effective amount of an antibody-drug conjugate as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Fig. 1A-B show the heavy and light chain CDRs (AbM definition; marked in bold) of the mouse anli-P'l’KV antibody, 13G11, ported onto the selected human VH and VL germline sequences to generate humanized sequence Hl (SEQ ID NO: 30) (Fig. 1A) and humanized sequence LI (SEQ ID NO: 34) (Fig. IB), respectively.

[0029] Fig. 2A-B show dose -response curves for binding to human PTK7 (Fig. 2A) and cynomolgus monkey PTK7 (Fig. 2B) for chimeric and selected humanized anti-PTK7 antibodies based on antibody 13G11. Positive control cofetuzumab (vl9287) and negative control palivizumab (v22277) are also shown.

[0030] Fig.3A-B show the heavy and light chain CDRs (AbM definition; marked in bold) of the mouse anti-PTK7 antibody, 14D08, ported onto the selected human VH and VL germlines to generate humanized sequence Hl (SEQ ID NO: 58) (Fig. 3A) and humanized sequence LI (SEQ ID NO: 62) (Fig.3B), respectively.

[0031] Fig. 4A-B show dose -response curves for binding to human PTK7 (Fig. 4A) and cynomolgus monkey PTK7 (Fig. 4B) for chimeric and selected humanized anti-PTK7 antibodiesbased on antibody 14D08. Positive control cofetuzumab (vl9287) and negative control palivizumab (v22277) are also shown.

[0032] Fig. 5A-D show binding of the humanized anti-PTK7 antibodies v39115 and v39133 to human PTK7 expressed on CHO-S cells (Fig. 5A); to cynomolgus monkey PTK7 expressed on CHO-S cells (Fig. 5B); to endogenously expressed PTK7 on HCC1569 breast cancer cells (Fig.5C), and to endogenously expressed PTK7 on H446 lung cancer cells (Fig. 5D). Positive control cofetuzumab (vl9287), a high-affinity monospecific anti-PTK7 antibody (v38332) and negative control palivizumab (v22277) are also shown.

[0033] Fig. 6A-B show internalization of anti-PTK7 antibodies, v39115, v39133, v38332 and cofetuzumab, into HCC1569 cells (Fig. 6A) and H446 cells (Fig. 6B) following 10 nM treatment with antibody for 4 hours. Negative control palivizumab is also shown.

[0034] Fig.7 shows the results of non-reducing (NR) and reducing (R) capillary electrophoresis analysis by Caliper for the biparatopic anli-PTK7 antibody variant v39794.

[0035] Fig. 8 shows the UPLC-SEC profile for the biparatopic anti-PTK7 antibody variant v39794.

[0036] Fig. 9A-B show the LC-MS profile for the biparatopic anti-PTK7 antibody variant v39794. The region of the MS spectrum showing full-size antibody peaks is shown in Fig.9A, and the region of the MS spectrum showing half-antibody peaks is shown in Fig. 9B.

[0037] Fig. 10A-B show binding of the biparatopic anti-PTK7 antibody variant, v39794, to endogenously expressed PTK7 on HCC1569 breast cancer cells (Fig. 10A) and on H446 lung cancer cells (Fig. 10B). Positive control cofetuzumab (vl9287), a high-affinity monospecific anti-PTK7 antibody (v38332) and negative control palivizumab (v22277) are also shown.

[0038] Fig. 11A-B show binding of the anti-PTK7 biparatopic antibody variant, v39794, to human PTK7 expressed on CHO-S cells (Fig. 11A), and to cynomolgus monkey PTK7 expressed on CHO-S cells (Fig. 11B). Positive control cofetuzumab (vl9287), a high-affinity monospecific anli-PTK7 antibody (v38332) and negative control palivizumab (v22277) are also shown.

[0039] Fig. 12A-B show internalization of the anti-PTK7 biparatopic antibody variant, v39794, the anti-PTK7 monospecific antibody, v38332, and cofetuzumab, into HCC1569 cells (Fig. 12A) and H446 cells (Fig. 12B) following 10 nM treatment with antibody for 4 hours. Negative control palivizumab is also shown.

[0040] Fig. 13A-B show penetration of anti-PTK7 biparatopic antibody variant, v39794, and anti-PTK7 monospecific antibodies, v39133, v39115 and v41357 into H446 cell line spheroids (Fig. 13A) and HCT116 cell line spheroids (Fig. 13B). Positive control cofetuzumab and negative control palivizumab are also shown.

[0041] Fig. 14A-C show the results of an assessment of cytotoxicity of the anti-PTK7 biparatopic ADC (v38794-DL-l DAR 8) and the anti-PTK7 monospecific ADCs (v41357-DL-l DAR 8, v38115-DL-l DAR 8 and v38133-DL-l DAR 8) in 2D monolayers of H446 cells (Fig.14A), DU4475 cells (Fig. 14B), and AsPC-1 cells (Fig. 14C). ADCs comprising positive control cofetuzumab and negative control palivizumab each conjugated to DL-1 at DAR 8 are also shown.

[0042] Fig. 15A-D show the results of an assessment of cytotoxicity of the anti-PTK7 biparatopic ADC (v38794-DL-l DAR 8) and the anti-PTK7 monospecific ADCs (v41357-DL-l DAR 8, V38115-DL-1 DAR 8 and v38133-DL-l DAR 8) in 3D spheroids of HCC1569 cells (Fig.15A), H446 cells (Fig. 15B), DU4475 cells (Fig. 15C), and AsPC-1 cells (Fig. 15D). ADCs comprising positive control cofetuzumab and negative control palivizumab each conjugated to DL-1 at DAR 8 are also shown.

[0043] Fig.16A-B shows bystander activity of anti-PTK7 biparatopic ADC (v38794-DL-l DAR 8), or anti-PTK7 monospecific ADC (v41357-DL-l DAR 8) against PTK7-low SK-BR-3 cell line (Fig. 16A) and against PTK7-high H446 cell line (Fig. 16B). Negative control palivizumab-DL-1 DAR 8 is also shown.

[0044] Fig. 17 shows serum concentrations of anti-PTK7 biparatopic ADC (v39794-DL-l DAR 8), anti-PTK7 monospecific ADC (v38332-DL-l DAR 8) and palivizumab-DL-1 (DAR 8) ADC, each dosed at 6 mg / kg, and cofetuzumab pelidotin, dosed at 2.5 mg / kg, in the NCI-H292 CDX model.

[0045] Fig. 18 shows the results of an in vivo efficacy study of the anti-PTK7 biparatopic ADC (v39794-DL-l DAR 8), anti-PTK7 monospecific ADC (v41357-DL-l DAR 8) and palivizumab-DL-1 (DAR 8) ADC, each dosed at 6 mg / kg, and cofetuzumab pelidotin, dosed at 2.5 mg / kg, in the NCI-H292 CDX model of human lung cancer.

[0046] Fig. 19A-K show the results of an in vivo efficacy study of anti-PTK7 biparatopic ADCs (v39794-DL-l DAR 8 and v39794-DL-l DAR 4), anti-PTK7 monospecific ADC (v41357-DL-l DAR 8) and palivizumab-DL-1 (DAR 8) ADC, each dosed at 6 mg / kg, and cofetuzumab pelidotin, dosed at 2.5 mg / kg, in the triple negative breast cancer PDX models, BR9457 (Fig. 19A), BR5017 (Fig. 19B), BR0438 (Fig. 19C), BR5013 (Fig. 19D), and in the non-small cell lung cancer PDX models, LU6913 (Fig. 19E), LU1235 (Fig. 19F), LU1215 (Fig. 19G), LU6911 (Fig. 19H), LU0299 (Fig. 191), LU6408 (Fig. 19J) and LU0884 (Fig. 19K).

[0047] Fig.20 shows the Membrane Proteome Array screen binding data for the biparatopic anti-PTK7 antibody variant, v39794, with validated target complexes highlighted.

[0048] Fig.21A-D show the validation data from the the Membrane Proteome Array evaluation of the biparatopic anti-PTK7 antibody variant, v39794, binding to GRIN1-GRIN3B and GRM5-ADORA2A complexes in pancellular cell types (Fig. 21A & B) and in extracellular cell types (Fig. 21C & D).

[0049] Fig. 22A-D show the results of an assessment of cytotoxicity of the anti-PTK7 biparatopic ADC (v38794-DL-2 DAR 4), and the anti-PTK7 monospecific ADCs (v41357-DL-2 DAR 4, v38115-DL-2 DAR 4 and v38133-DL-2 DAR 4) in 2D monolayers of HCC1569 cells (Fig. 22A), DU4475 cells (Fig. 22B), TOV-21G cells (Fig. 22C) and AsPC-1 cells (Fig. 22D).ADCs comprising positive control cofetuzumab and negative control palivizumab each conjugated to DL-2 at DAR 4 are also shown.

[0050] Fig. 23A-B show the intracellular (Fig. 23A) and extracellular (Fig. 23B) payload quantitation in PTK7-expressing cell line HCC1569 after a 24-hour treatment with 20 nM of the anti-PTK7 biparatopic ADC (v38794-DL-2 DAR 4), and the anti-PTK7 monospecific ADCs (v41357-DL-2 DAR 4, v38115-DL-2 DAR 4 and v38133-DL-2 DAR 4). ADCs comprisingpositive control cofetuzumab and negative control palivizumab each conjugated to DL-2 at DAR 4 are also shown.

[0051] Fig. 24 shows the CDR sequences of humanized versions of the VH and VL sequences of the anli-P'I’K? antibodies, v39093 and v39090.

[0052] Fig. 25 shows the first dose serum total anlibody concentrations for the anli-P'I’K? monospecific ADC (v41357-DL-l DAR 8) and the anti-PTK7 biparatopic ADC (v43464-DL-l DAR 8) in male cynomolgus monkeys.

[0053] Fig. 26A-B shows internalization of the anti-PTK7 biparatopic anlibody v39794 and the anti-PTK7 monospecific antibodies, v41357, v44734 and v44733, into PTK7-expressing cell lines; HCC1569 breast cancer cells (Fig. 26A), and H446 lung cancer cells (Fig. 26B). Positive control cofetuzumab (vl9287) and negative control palivizumab (v21995) are also shown.

[0054] Fig. 27 shows penetration of the anti-PTK7 biparatopic antibody v39794 and the anti-PTK7 monospecific antibodies, v41357, v44734 and v44733, into H446 cell line spheroids. Positive control cofetuzumab (vl9287) and negative control palivizumab (v21995) are also shown.

[0055] Fig. 28A-F show the results of an in vivo efficacy study of anti-PTK7 biparatopic ADC v39794-DL-l DAR 8 in various CDX models; NCI-H2228 lung cancer (Fig.28A), DU4475 breast cancer (Fig. 28B), NCI-H358 lung cancer (Fig. 28C), NCI-H1373 lung cancer (Fig. 28D), NCI-11441 lung cancer (Fig. 28E) and LU99 lung cancer (Fig. 28F). The ADC palivizumab-DL-1 (DAR 8) was included as a negative control. In some models the monospecific anti-PTK7 ADC v38332-DL-l (DAR 8) or the ADC cofetuzumab pelidotin were included as targeted controls.

[0056] Fig. 29A-B show the serum concentrations of anti-PTK7 biparatopic antibody v43464 (wild-type Fc) (Fig. 29A) and anti -PTK7 biparatopic antibody v45116 (YTE mutations) (Fig.29B) in male Tg32 SCID mice with and without IVIG preload.

[0057] Fig. 30A-B show the unconjugated payload (Compound 1) (Fig. 30A) and antibody-conjugated payload (Fig. 30B) serum concentrations after administration of 60 mg / kg of the anti-PTK monospecific ADC v41357-DLl (DAR 8) or the anti-PTK7 biparatopic ADC v43464-DLl (DAR 8) in cynomolgus monkeys.DETAILED DESCRIPTION

[0058] The present disclosure relates to antibody constructs that bind to human PTK7 (“anti-PTK7 antibody constructs”). The anti-PTK7 antibody constructs may be monospecific, bispecific or multispecific. In certain embodiments, the anti-PTK7 antibody constructs are bispecific. Bispecific anti-PTK7 antibody constructs may bind to PTK7 and another non-PTK7 protein, or they may bind to two different epitopes on PTK7 (i.e. they may be biparatopic). In some embodiments, the anti-PTK7 antibody constructs are biparatopic.

[0059] The present disclosure also relates to antibody-drug conjugates (ADCs) comprising an anti-PTK7 antibody construct conjugated to one or more drug moieties. Certain embodiments relate to ADCs comprising a biparatopic anti-PTK7 antibody construct conjugated to one or more drug moieties. The anti-PTK7 antibody constructs and ADCs may find use, for example, as therapeutics or diagnostics. Certain embodiments of the present disclosure relate to therapeutic or diagnostic methods and uses of the anti-PTK7 antibody constructs and ADCs, for example, in the treatment or diagnosis of cancer.Definitions

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0061] As used herein, the term “about” refers to an approximately + / -10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0062] The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0063] As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. The term “consisting essentially of’ when used herein in connection with a composition, use or method, denotes that additional elements and / or methodsteps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term “consisting of’ when used herein in connection with a composition, use or method, excludes the presence of additional elements and / or method steps. A composition, use or method described herein as comprising certain elements and / or steps may also, in certain embodiments consist essentially of those elements and / or steps, and in other embodiments consist of those elements and / or steps, whether or not these embodiments are specifically referred to.

[0064] A “complementarity determining region” or “CDR” is an amino acid sequence that contributes to antigen-binding specificity and affinity. “Framework” regions (FR) can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen-binding region and an antigen. From N-terminus to C-terminus, both the light chain variable region (VL) and the heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. Often, the three heavy chain CDRs and the three light chain CDRs are required to bind antigen. However, in some instances, even a single variable domain can confer binding specificity to the antigen. Furthermore, as is known in the art, in some cases, antigen-binding may also occur through a combination of a minimum of one or more CDRs selected from the VH and / or VL domains, for example HCDR3.

[0065] A number of different definitions of the CDR sequences are in common use, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), by Chothia et al. (1987, J Mol Biol, 196:901-917), as well as the IMGT, AbM (University of Bath) and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745) definitions. By way of example, CDR definitions according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Accordingly, as would be readily apparent to one skilled in the art, the exact numbering and placement of CDRs may differ based on the numbering system employed. However, it is to be understood that the disclosure herein of a VH sequence includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems. Similarly, disclosure herein of a VL sequenceincludes the disclosure of the associated (inherent) light chain CDRs (LCDRs) as defined by any of the known numbering systems.Table 1: Common CDR Definitions1Definition Heavy Chain Light ChainCDR12CDR2 CDR3 CDR1 CDR2 CDR3 Kabat H31-H35B H50-H65 H95-H102 L24-L34 L50-L56 L89-L97 Chothia H26-H32, H52-H56 H95-H102 L24-L34 L50-L56 L89-L97 H33 orH34IMGT H26-H33, H51-H57 H93-H102 L27-L32 L50-L52 L89-L97 H34, H35,H35A orH35BAbM H26-H35B H50-H58 H95-H102 L24-L34 L50-L56 L89-L97 Contact H30-H35B H47-H58 H93-H101 L30-L36 L46-L55 L89-L961Either the Kabat or Chothia numbering system may be used for HCDR2, HCDR3 and the light chain CDRs for all definitions except Contact, which uses Chothia numbering2Using Kabat numbering. The position in the Kabat numbering scheme that demarcates the end of the Chothia and IMGT CDR-H1 loop varies depending on the length of the loop because Kabat places insertions outside of those CDR definitions at positions 35 A and 35B. However, the IMGT and Chothia CDR-H1 loop can be unambiguously defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0066] The term “identical” in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (for example, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity, over a specified region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the commonly used sequence comparison algorithms as known to persons of ordinary skill in the art or by manual alignment and visual inspection. For sequence comparison, typically test sequences are compared to a designated reference sequence. When using a sequence comparison algorithm, test andreference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0067] A “comparison window” refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions which may be, for example, from about 10 to 600 contiguous amino acid or nucleotide positions, or from about 10 to about 200, or from about 10 to about 150 contiguous amino acid or nucleotide positions over which a test sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. AppL Math., 2:482c; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computerized implementations of these algorithms (for example, GAP, BESTFIT, FASTA or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the website for the National Center for Biotechnology Information (NCBI).

[0068] The term “subject,” as used herein, refers to an animal, in some embodiments a mammal, which is the object of treatment, observation or experiment. The animal may be a human, a nonhuman primate, a companion animal (for example, dog, cat, or the like), farm animal (for example, cow, sheep, pig, horse, or the like) or a laboratory animal (for example, rat, mouse, guinea pig, non-human primate, or the like). In certain embodiments, the subject is a human.

[0069] Where a range of values is provided herein, for example where a value is defined as being “between” an upper limit value and a lower limit value, it is understood that the range encompasses both the upper limit value and the lower limit value as well as each intervening value.

[0070] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use or composition disclosed herein, and vice versa.

[0071] Particular features, structures and / or characteristics described in connection with an embodiment disclosed herein may be combined with features, structures and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

[0072] It is also to be understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in an alternative embodiment. For example, where a list of options is presented for a given embodiment or claim, it is to be understood that one or more option may be deleted from the list and the shortened list may form an alternative embodiment, whether or not such an alternative embodiment is specifically referred to.ANTI-PTK7 ANTIBODY CONSTRUCTS

[0073] The present disclosure relates to antibody constructs that bind to human Protein Tyrosine Kinase 7 (PTK7). In this context, the term “anlibody construct” refers to a polypeptide or a set of polypeptides that comprises one or more antigen-binding domains, where each of the one or more antigen-binding domains binds to an epitope or antigen. Where the anlibody construct comprises two or more antigen-binding domains, each of the antigen-binding domains may bind the same epitope or antigen (i.e. the anlibody construct is monospecific) or they may bind to different epitopes or antigens (i.e. the antibody construct is bispecific or multispecific). In certain embodiments, the antibody construct may bind to two different epitopes on PTK7 (i.e. the antibody construct is biparatopic). The antibody construct may optionally further comprise a scaffold and the one or more antigen-binding domains can be fused or covalently attached to the scaffold, optionally via a linker, as described herein.

[0074] In accordance with the present disclosure, the anti-PTK7 anlibody construct comprises at least one antigen-binding domain that binds to human PTK7. In certain embodiments, the antigen-binding domain specifically binds to human PTK7. By “specifically binds” to human PTK7, it is meant that the antigen-binding domain of the antibody construct binds to human PTK7 with a KD of 250nM or less. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure may also be capable of binding to PTK7 from one or more non-human species. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure are capable of binding to both human PTK7 and cynomolgus monkey PTK7.

[0075] Human PTK7 is also known as “Colon Carcinoma Kinase 4” or CCK4. The protein sequences of PTK7 from various sources are known in the art and readily available from publicly accessible databases, such as GenBank or UniProtKB. Examples of human PTK7 sequences include for example those provided in GenBank under NCBI Reference Numbers: NP_002812.2, KAI2542404.1 and AAH71557.1. An exemplary human PTK7 protein sequence is provided in Table 2 as SEQ ID NO: 1 (NCBI Reference Number: NP_002812.2). An exemplary cynomolgus monkey PTK7 protein sequence (NCBI Reference Number: XP_005552998) is also provided in Table 2 as SEQ ID NO: 95.Table 2: Amino Acid Sequences of Human and Cynomolgus Monkey PTK7Species Sequence SEQ ID NOHuman MGAARGSPARPRRLPLLSVLLLPLLGGTQTAIVFIKQPSSQDALQ 1 GRRALLRCEVEAPGPVHVYWLLDGAPVQDTERRFAQGSSLSFA AVDRLQDSGTFQCVARDDVTGEEARSANASFNIKWIEAGPVVL KHPASEAEIQPQTQVTLRCHIDGHPRPTYQWFRDGTPLSDGQSN HTVSSKERNLTLRPAGPEHSGLYSCCAHSAFGQACSSQNFTLSIA DESFARVVLAPQDVVVARYEEAMFHCQFSAQPPPSLQWLFEDE TPITNRSRPPHLRRATVFANGSLLLTQVRPRNAGIYRCIGQGQRG PPIILEATLHLAEIEDMPLFEPRVFTAGSEERVTCLPPKGLPEPSV WWEHAGVRLPTHGRVYQKGHELVLANIAESDAGVYTCHAANL AGQRRQDVNITVATVPSWLKKPQDSQLEEGKPGYLDCLTQATP KPTVVWYRNQMLISEDSRFEVFKNGTLRINSVEVYDGTWYRC MSSTPAGSIEAQARVQVLEKLKFTPPPQPQQCMEFDKEATVPCS ATGREKPTIKWERADGSSLPEWVTDNAGTLHFARVTRDDAGNY TCIASNGPQGQIRAHVQLTVAVFITFKVEPERTTVYQGHTALLQC EAQGDPKPLIQWKGKDRILDPTKLGPRMHIFQNGSLVIHDVAPE DSGRYTCIAGNSCNIKHTEAPLYVVDKPVPEESEGPGSPPPYKMI QTIGLSVGAAVAYIIAVLGLMFYCKKRCKAKRLQKQPEGEEPEMECLNGGPLQNGQPSAEIQEEVALTSLGSGPAATNKRHSTSDKMHSpecies Sequence SEQ ID NO FPRSSLQPITTLGKSEFGEVFLAKAQGLEEGVAETLVLVKSLQSK DEQQQLDFRRELEMFGKLNHANVVRLLGLCREAEPHYMVLEY VDLGDLKQFLRISKSKDEKLKSQPLSTKQKVALCTQVALGMEH LSNNRFVHKDLAARNCLVSAQRQVKVSALGLSKDVYNSEYYH FRQAWVPLRWMSPEAILEGDFSTKSDVWAFGVLMWEVFTHGE MPHGGQADDEVLADLQAGKARLPQPEGCPSKLYRLMQRCWAL SPKDRPSFSEIASALGDSTVDSKPCynomolgus MGATRGSLARPRRLPLLSVLLLPLLGGTQAAIVFIKQPSSQDALQ 95 Monkey GRRALLRCEVEAPGLVHVYWLLDGAPVQDTERRFTQGSSLSFA AVDRLQDSGTFQCVARDDVTGEEARSANASFNIKWIEAGPVVL KHPASEAEIQPQTQVTLRCHIDGHPRPTYQWFRDGTPLSDGQSN NTVSSKERNLTLRPAGPEHSGLYSCCAHNAFGQACSSQNFTLSIA DESFARVVLAPQDVIVARNEEAMFHCQFSAQPPPNLQWLFEDET PITNRSRPPHLRRATVFANGSLLLTQVRPRNAGVYRCIGQGQRG PPVILEATLHLAEIEDMPLFEPRVFTAGSEERVTCLPPKGLPEPSV WWEHAGVRLPTHGRVYQKGHELVLASIAESDAGVYTCHAANL AGQRRQDVNITVATVPTWLKKPQDSQLEEGKPGYLHCLTQATP KPTVVWYRNQMLISEDSRFEVFKNGTLRINNVEVYDGTWYRC VSSTPAGSIEAQARVQVLEKLKFTPPPQPQQCMEFDKEATVPCS ATGREKPTIKWERADGSSLPEWVTDNAGTLHFARVTRDDAGNY TCIASNGPQGQIRAHVQLTVAVFITFKVEPERTTVYQGHTALLQC EAQGDPKPLIQWKGKDRILDPTKLGPRMHIFQNGSLVIHDVAPE DSGRYTCIAGNSCNIKHTEAPLYVVDKPVPEESEGPGSPPPYKMI QTIGLSVGAAVAYIIAVLGLMFYCKKRCKAKRLQKQPEGEEPEM ECLNGGPLQNGQPSAEIQEEVALTSLGSGPAATNKRHSTSDKMH FPRSSLQPITTLGKSEFGEVFLAKAQGLEEGVAETLVLVKSLQSK DEQQQLDFRRELEMFGKLNHANVVRLLGLCREAEPHYMVLEY VDLGDLKQFLRISKSKDEKLKSQPLSTKQKVALCTQVALGMEH LSNNRFVHKDLAARNCLVSAQRQVKVSALGLSKDVYNSEYYH FRQAWVPLRWMSPEAILEGDFSTKSDVWAFGVLMWEVFTHGE MPHGGQADDEVLADLQAGKARLPQPEGCPSKLFRLMQRCWAL SPKDRPSFSEIASTLGDSPVDSKP

[0076] Binding of an antigen-binding domain to a target antigen or epitope, such as human or cynomolgus PTK7, may be measured, for example, through an enzyme-linked immunosorbent assay (ELISA), a surface plasmon resonance (SPR) technique (employing, for example, a BIAcore® instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), a solution-based kinetic exclusion assay (KinExA™), flow cytometry or a traditional binding assay (Heeley, 2002, Endocr Res, 28:217-229).

[0077] In certain embodiments, binding of an antibody construct to human PTK7 may be defined by a dissociation constant. The term “dissociation constant ( D or Kd),” as used herein, is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction, such as antibody-antigen interactions. The KD measures the propensity of two proteins complexed together (e.g. AB) to dissociate reversibly into constituent components (A+B) and is defined as the ratio of the dissociation rate constant, also called the “off-rate (kon),” to the association rate constant, or “on-rate (kon)”. Thus, KD equals kOff / kOn and is expressed as a molar concentration (M). It follows that the smaller the KD, the stronger the affinity of binding, and thus a decrease in KD indicates an increase in affinity. KD between an antibody and its antigen can be determined using methods well established in the art. One method for determining KD is by using surface plasmon resonance (SPR), typically using a biosensor system such as a BIAcore® system. Isothermal titration calorimetry (ITC) is another method that can be used to measure KD. The Octet™ system or KinExA™ system may also be used to measure the affinity of antibodies for a target antigen. As is known in the art the numerical value of the KD obtained may vary depending on how it is tested. For example, the numerical value of KD when measured in a cell-based assay may be affected by the expression level of the target antigen (in this case, PTK7) in the cell line, format of the antibody construct (i.e. monovalent or bivalent) and / or type of assay. The data provided in the Examples illustrate this general point.

[0078] In certain embodiments, binding of an anti-PTK7 antibody construct to human PTK7 may be defined by a dissociation constant (KD) of <500 nM, for example, <250 nM, <100 nM, <50 nM, <25 nM or <10 nM, as determined by SPR. In certain embodiments, specific binding of an antibody construct to human PTK7 may be defined by a KD of 10’6M or less, for example, 10’7M or less, or 10’8M or less, as determined by SPR. In some embodiments, specific binding of an antibody construct to human PTK7 may be defined by a KD between about 50 nM and about 10 nM, for example, between about 25 nM and about 10 nM, as determined by SPR.Antigen-Binding Domains

[0079] The anti-PTK7 antibody constructs of the present disclosure comprise at least one antigen-binding domain that is capable of binding to human PTK7 (a “PTK7 antigen-binding domain”). The at least one PTK7 antigen-binding domain typically is an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of an antigen-bindingantibody fragment include, but are not limited to, a Fab fragment, a Fab’ fragment, a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb).

[0080] A “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI) along with the variable domains of the light and heavy chains (VL and VH, respectively). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region. A Fab fragment may also be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0081] An “scFv” includes a heavy chain variable domain (VH) and a tight chain variable domain (VL) of an antibody in a single polypeptide chain. The scFv may optionally further comprise a polypeptide tinker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding. For example, an scFv may include a VL connected from its C-terminus to the N-terminus of a VH by a polypeptide tinker. Alternately, an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide linker (see review in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994)).

[0082] An “sdAb” format refers to a single immunoglobulin domain. The sdAb may be, for example, of camelid origin. Camelid antibodies lack light chains and their antigen-binding sites consist of a single domain, termed a “VHH.” An sdAb comprises three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and easy to express, for example, as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1): 13-22).

[0083] In those embodiments in which the anti-PTK7 antibody constructs comprise two or more antigen-binding domains, each additional antigen-binding domain may independently be an immunoglobulin-based domain, such as an antigen-binding antibody fragment as described above, or a non-immunoglobulin-based domain, such as a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule capable of specifically binding to its target, for example, anatural or engineered ligand. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, fynomers, affimers, alphabodies, DARPins and avimers. In certain embodiments, the anti-PTK7 antibody constructs comprise two or more antigen-binding domains and each additional antigen-binding domain is an immunoglobulin-based domain, such as an antigen-binding antibody fragment. In certain embodiments, the anti-PTK7 antibody construct comprises two PTK7 antigen-binding domains, which may be the same or different, and in which both PTK7 antigen-binding domains are antigen-binding antibody fragments.

[0084] The present disclosure describes the identification of two antibodies that bind human PTK7 and which also bind cynomolgus monkey PTK7 (variants v39093 and v39090), as well as representative humanized versions of each of these antibodies (see Examples 1, 3 and 4).

[0085] The VH and VL sequences of the anti-PTK7 antibodies v39093 and v39090 are provided in Table 1.1, and the CDR sequences are provided in Tables 3.1 and 4.1, respectively. The different humanized heavy (VH) and light (VL) chains of antibody v39093 are provided in Table 3.3 and the CDR sequences are provided in Fig. 24. The different humanized heavy (VH) and light (VL) chains of antibody v39090 are provided in Table 4.3 and the CDR sequences are provided in Fig.24.

[0086] In certain embodiments, the anti-PTK7 antibody constructs comprise a PTK7 antigenbinding domain comprising:(a) the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36; or(b) the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64,

[0087] where the CDR sequences are as defined by one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0088] In certain embodiments, the anti-PTK7 antibody constructs comprise a PTK7 antigenbinding domain comprising heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) and light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of any one of variants v39093, v39115, v39090 or v39133, as defined by one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems. In some embodiments, the anti-PTK7 antibody constructs comprise a PTK7 antigenbinding domain comprising heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) and light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of variant v39115 or v39133, as defined by one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0089] One skilled in the art will appreciate that a limited number of amino acid substitutions may be introduced into the CDR sequences or into the VH or VL sequences of known antibodies without the antibody losing its ability to bind its target. Candidate amino acid substitutions may be identified by computer modeling or by art-known techniques such as alanine scanning, with the resulting variants being tested for binding activity by standard techniques. Typically, such amino acid substitutions are conservative amino acid substitutions. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure may comprise a PTK7 antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v39093, v39115, v39090 or v39133, where the variant comprises between 1 and 6 amino acid substitutions across the set of CDRs (i.e. the CDRs may be modified by up to 6 amino acid substitutions with any combination of the six CDRs being modified), and where the antigen-binding domain retains the ability to bind human PTK7. In some embodiments, the anti-PTK7 antibody constructs comprise a PTK7 antigen-binding domain that comprises a variant of the set of CDR sequences of any one of variants v39093, v39115, v39090 or v39133, where the variant comprises between 1 and 5 amino acid substitutions, between 1 and 4 amino acid substitutions, between 1 and 3 amino acid substitutions, between 1 and 2 amino acid substitutions, or 1 amino acid substitution, across the set of CDRs, and where the antigen-binding domain retains the ability to bind human PTK7.

[0090] In certain embodiments, the anti-PTK7 antibody constructs comprise a PTK7 antigenbinding domain comprising:(a) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 having a sequence selected fromthe amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16, 18, 88 or 101; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24, 26 or 89, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; or(b) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 41, 44, 46, 48, 92, 93 or 94; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 39, 42, 49, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57.

[0091] In certain embodiments, the anti-PTK7 antibody constructs comprise a PTK7 antigenbinding domain comprising:(a) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; or(b) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected fromthe amino acid sequences as set forth in any one of SEQ ID NOs: 38, 41, 44, 46 or 48; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 39, 42 or 49; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57; or an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57. In certain embodiments, the anti-PTK7 antibody constructs comprise a PTK7 antigenbinding domain comprising:(a) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; or(b) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected fromthe amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 2 or 4, where the PTK7 antigen-binding domain retains the ability to bind human PTK7. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 3 or 5, where the PTK7 antigen-binding domain retains the ability to bind human PTK7.

[0094] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33, where the antigen-binding domain retains the ability to bind human PTK7. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36, where the antigen-binding domain retains the ability to bind human PTK7.

[0095] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forthin any one of SEQ ID NOs: 31, 32 or 33, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36, where the antigen-binding domain retains the ability to bind human PTK7. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, where the antigen-binding domain retains the ability to bind human PTK7. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34, where the antigen-binding domain retains the ability to bind human PTK7. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34, where the antigenbinding domain retains the ability to bind human PTK7.

[0098] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61, where the antigen-binding domain retains the ability to bind human PTK7. In certain embodiments, the anti-PTK7 antibody constructs of the presentdisclosure comprise a PTK7 antigen-binding domain that comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64, where the antigen-binding domain retains the ability to bind human PTK7.

[0099] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64, where the antigen-binding domain retains the ability to bind human PTK7.

[0100] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58, where the antigen-binding domain retains the ability to bind human PTK7. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62, where the antigen-binding domain retains the ability to bind human PTK7.

[0101] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62, where the antigenbinding domain retains the ability to bind human PTK7.

[0102] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises:(a) a VH sequence comprising the sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33, and a VL sequence comprising the sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36, or(b) a VH sequence comprising the sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61, and a VL sequence comprising the sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64.

[0103] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise a PTK7 antigen-binding domain that comprises:(a) a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, and a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34; or(b) a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58, and a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62.Formats

[0104] The anti-PTK7 antibody constructs of the present disclosure may have various formats. The minimal component of the anti-PTK7 antibody construct is an antigen-binding domain that binds to human PTK7. The anti-PTK7 antibody constructs may further optionally comprise one or more additional antigen-binding domains and / or a scaffold. In those embodiments in which the anti-PTK7 antibody construct comprises two or more antigen-binding domains, each additionalantigen-binding domain may independently bind to the same epitope within human PTK7, may bind to a different epitope within human PTK7, or may bind to a different antigen. Thus, the anti-PTK7 antibody construct may be, for example, monospecific, biparatopic, bispecific or multispecific.

[0105] In certain embodiments, the anti-PTK7 antibody construct comprises at least one antigenbinding domain that binds to human PTK7 and a scaffold, where the antigen-binding domain is operably linked to the scaffold. The term “operably linked,” as used herein, means that the components described are in a relationship permitting them to function in their intended manner. Examples of suitable scaffolds are described below.

[0106] In certain embodiments, the anti-PTK7 antibody construct comprises two antigen-binding domains optionally operably linked to a scaffold. In some embodiments, the anti-PTK7 antibody construct may comprise three or four antigen-binding domains and optionally a scaffold. In these formats, when comprising a scaffold, at least a first antigen-binding domain is operably linked to the scaffold and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or to the first antigen-binding domain or, when more than two antigenbinding domains are present, to another antigen-binding domain.

[0107] Anti-PTK7 antibody constructs that lack a scaffold may comprise a single antigenbinding domain in an appropriate format, such as an sdAb, or they may comprise two or more antigen-binding domains optionally operably linked by one or more linkers. In such anti-PTK7 antibody constructs, the antigen-binding domains may be in the form of scFvs, Fabs, sdAbs, or a combination thereof. For example, using scFvs as the antigen-binding domains, formats such as a tandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker. scFvs may also be used to construct diabody formats, which comprise two scFvs connected by a short linker (usually about 5 amino acids in length). The restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs may be further stabilized by inclusion of an interdomain disulfide bond. For example, a disulfide bond may be introduced between VL and VH through introduction of an additional cysteine residue in each chain (for example, at position 44 in VH and position 100 in VL) (see, for example, Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may beintroduced between two VHs to provide a construct having a DART format (see, for example, Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0108] Similarly, formats comprising two sdAbs, such as VHs or VHHs, connected together through a suitable linker may be employed in some embodiments. Other examples of anti-PTK7 antibody construct formats that lack a scaffold include those based on Fab fragments, for example, Fab2 and F(ab’)2 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.

[0109] Combinations of antigen-binding domains in different forms may also be employed to generate alternative scaffold-less formats. For example, an scFv or a sdAb may be fused to the C-terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv / sdAb) construct.

[0110] In certain embodiments, the anti-PTK7 antibody construct may be in an antibody format that is based on an immunoglobulin (Ig). In certain embodiments, the anti-PTK7 antibody construct may be based on an IgG class immunoglobulin, for example, an IgGl, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the anti-PTK7 antibody construct may be based on an IgGl immunoglobulin. In the context of the present disclosure, when an anti-PTK7 antibody construct is based on a specified immunoglobulin isotype, it is meant that the anti-PTK7 antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-PTK7 antibody construct based on a given Ig isotype may comprise at least one antigen-binding domain operably linked to an Ig scaffold, where the scaffold comprises an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype. It is to be understood that the anti-PTK7 antibody constructs may also comprise hybrids of isotypes and / or subclasses in some embodiments. It is also to be understood that the Fc region and / or hinge region may optionally be modified to impart one or more desirable functional properties as is known in the art.

[0111] In some embodiments, the anti-PTK7 antibody constructs may be derived from two or more immunoglobulins that are from different species, for example, the anti-PTK7 antibody construct may be a chimeric antibody or a humanized antibody. The terms “chimeric antibody”and “humanized antibody” both refer generally to antibodies that combine immunoglobulin regions or domains from more than one species.

[0112] A “chimeric antibody” typically comprises at least one variable domain from a nonhuman antibody, such as a rabbit or rodent (for example, murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison etal., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U. S. Patent No. 4,816,567.

[0113] A “humanized antibody” is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by residues from a hypervariable region (CDR) of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, having the desired specificity and affinity for a target antigen. This technique for creali ng humanized antibodies is often referred to as “CDR grafting.”

[0114] In some instances, additional modifications may be made to a humanized antibody to further refine antibody performance. For example, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or the humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. In general, a variable domain in a humanized antibody will comprise all or substantially all of the hypervariable regions from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence. Humanized antibodies are described in more detail in Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596, for example.

[0115] A number of approaches are known in the art for selecting the most appropriate human frameworks into which to graft the non-human CDRs. Early approaches used a limited subset of well-characterised human antibodies, irrespective of the sequence identity to the non-human antibody providing the CDRs (the “fixed frameworks” approach). More recent approaches have employed variable regions with high amino acid sequence identity to the variable regions of thenon-human antibody providing the CDRs (“homology matching” or “best-fit” approach). An alternative approach is to select fragments of the framework sequences within each light or heavy chain variable region from several different human antibodies. CDR-grafting may in some cases result in a partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by back-mutating some of the residues of human origin to the corresponding non-human ones. Methods for preparing humanized antibodies by these approaches are well-known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0116] Alternatively, or in addition to, these traditional approaches, more recent technologies may be employed to further reduce the immunogenicity of a CDR-grafted humanized antibody. For example, frameworks based on human germline sequences or consensus sequences may be employed as acceptor human frameworks rather than human frameworks with somatic mutation(s). Another technique that aims to reduce the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues required for antigen-binding activity (the “SDRs”) are grafted into a human germline framework. This method improves the “humanness” (z.e. the similarity to human germline sequence) of the humanized antibody and thus may help reduce the risk of immunogenicity of the variable region. These techniques have been described in various publications (see, for example, Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872, and Kashmiri, et al., 2005, Methods, 36:25-34).

[0117] In certain embodiments, the anti-PTK7 antibody construct of the present disclosure comprises humanized antibody sequences, for example, one or more humanized variable domains (VH and / or VL). Non-limiting examples of humanized VH and VE sequences based on the anti-PTK7 antibodies v39093 and v39090 are described herein (see Examples and Tables 3.3 and 4.3).Scaffolds

[0118] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure comprise one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) may be in one or a combination of the forms described above (for example, scFvs, Fabs and / or sdAbs). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogues and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming “zipper” peptides derived from Jun and Fos, IgG CHI and CL domains or barnase-barstar toxins), cytokines, chemokines or growth factors. Other examples include antibodies based on the DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0119] A scaffold may be a peptide, polypeptide, polymer, nanoparticle or other suitable chemical entity. Where the scaffold is a polypeptide, each antigen-binding domain of the anti-PTK7 antibody construct may be linked to either the N- or C-terminus of the polypeptide scaffold. Anti-PTK7 antibody constructs comprising a polypeptide scaffold in which one or more of the antigen-binding domains are linked to a region other than the N- or C-terminus, for example, via the side chain of an amino acid with or without a linker, are also contemplated in certain embodiments.

[0120] In embodiments where the anti-PTK7 antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0121] A number of protein domains are known in the art that comprise selective pairs of two different polypeptides and may be used to form a scaffold. An example is leucine zipper domains such as Fos and Jun that selectively pair together (Kostelny, etal., J Immunol, 148:1547-53 (1992); Wranik, etal., J. Biol. Chem., 287: 43331-43339 (2012)). Other selectively pairing molecular pairs include, for example, the barnase-barstar pair (Deyev, et al., Nat Biotechnol, 21:1486-1492(2003)), DNA strand pairs (Chaudri, et al., FEBS Letters, 450(l-2):23-26 (1999)) and split fluorescent protein pairs (International Patent Application Publication No. WO 2011 / 135040).

[0122] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogues and derivatives, toxins, cytokines, chemokines and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Muller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154, and U. S. Patent Application Publication No.2009 / 0285816).

[0123] For example, fusing antigen-binding moieties such as scFvs, diabodies or single chain diabodies to albumin has been shown to improve the serum half-life of the antigen-binding moieties (Muller et al., ibid.). Antigen-binding moieties may be fused at the N- and / or C-termini of albumin, optionally via a linker. Derivatives of albumin in the form of heteromultimers that comprise two transporter polypeptides obtained by segmentation of an albumin protein such that the transporter polypeptides self-assemble to form quasi-native albumin have been described (see International Patent Application Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentation of albumin, the heteromultimer includes four termini and thus can be fused to up to four different antigen-binding moieties, optionally via linkers.

[0124] In certain embodiments, the anti-PTK7 antibody construct may comprise a protein scaffold. In some embodiments, the anti-PTK7 antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative. In some embodiments, the anti-PTK7 antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, for example, an IgG Fc region.Fc Regions

[0125] The terms “Fc region,” “Fc” or “Fc domain” as used herein refer to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0126] In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure may comprise a scaffold that is based on an immunoglobulin Fc region. The Fc region may be dimeric and composed of two Fc polypeptides or alternatively, the Fc region may be composed of a single polypeptide. In certain embodiments, the anti-PTK7 antibody constructs of the present disclosure may comprise a scaffold that is a dimeric Fc region.

[0127] An “Fc polypeptide” in the context of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association. When referring to the polypeptides forming a dimeric Fc region, the terms “first Fc polypeptide” and “second Fc polypeptide” may be used interchangeably provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0128] An Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, an Fc polypeptide of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0129] In some embodiments, the anti-PTK7 antibody construct may comprise a scaffold that is based on an IgG Fc region. In some embodiments, the anti-PTK7 antibody construct may comprise a scaffold that is based on a human IgG Fc region. In some embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on an IgGl Fc region. In some embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on a human IgGl Fc region.

[0130] In certain embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on an IgG Fc region, which is a homodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are the same.

[0131] In certain embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on an IgG Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and asecond Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are different. In some embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications.

[0132] In some embodiments, the anti-PTK7 antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain is an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, an “asymmetric amino acid modification” refers to a modification, such as a substitution or an insertion, in which an amino acid at a specific position on a first CH3 or CH2 sequence is different to the amino acid on a second CH3 or CH2 sequence at the same position. These asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.

[0133] In some embodiments, the anti-PTK7 antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, where the modified CH3 domain comprises one or more amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0134] Amino acid modifications that may be made to the CH3 domain of an Fc in order to promote formation of a heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 (“knobs into holes”), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”), Davis et al., 2010, ProtEng Des Sel, 23(4): 195-202 (strand exchange engineered domain (SEED) technology) and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab-arm exchange). Other examples includeapproaches combining positive and negative design strategies to produce stable asymmetrically modified Fc regions as described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication No. WO 2012 / 058768 or WO 2013 / 063702.

[0135] Table 3 provides the amino acid sequence of the human IgGl Fc sequence (SEQ ID NO: 100), corresponding to amino acids 231 to 447 of the full-length human IgGl heavy chain. The CH3 sequence comprises amino acids 341 to 447 of the full-length human IgGl heavy chain. Also shown in Table 3 are CH3 domain amino acid modifications that promote formation of a heterodimeric Fc as described in in International Patent Application Publication Nos. WO 2012 / 058768 and WO 2013 / 063702.

[0136] In certain embodiments, the anti-PTK7 antibody construct comprises a heterodimeric Fc scaffold having a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 3.Table 3: Human IgGl Fc Sequence1and CH3 Domain Amino Acid Modifications Promoting Heterodimer Formation APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 100)Variant No Chain Mutations1 A L351Y_F405A_Y407VB T366L_K392M_T394W2 A L351Y_F405A_Y407VB T366L_K392L_T394W3 A T350V_L351Y_F405A_Y407VB T350V_T366L_K392L_T394W4 A T350V_L351Y_F405A_Y407VB T350V_T366L_K392M_T394W5 A T350V_L351Y_S400E_F405A_Y407VB T350V_T366L_N390R_K392M_T394W1Sequence from positions 231-447 (EU numbering)

[0137] In some embodiments, the anti-PTK7 antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcRs) to the Fc, such as receptors of the FcyRI, FcyRII and FcyRIII subclasses.

[0138] In some embodiments, the anti-PTK7 antibody construct comprises a scaffold based on an IgG Fc having a modified CH2 domain, wherein the modification of the CH2 domain results in altered binding to one or more of the FcyRI, FcyRII and FcyRIII receptors.

[0139] A number of amino acid modifications to the CH2 domain that selectively alter the affinity of the Fc for different Fey receptors are known in the art. Amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding can each be useful in certain indications. For example, increasing binding affinity of an Fc for FcyRIIIa (an activating receptor) may result in increased antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell. Decreased binding to FcyRIIb (an inhibitory receptor) likewise may be beneficial in some circumstances. In certain indications, a decrease in, or elimination of, ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains comprising amino acid modifications that result in increased binding to FcyRIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all of the Fey receptors (“knock-out” variants) may be useful.

[0140] Examples of amino acid modifications to the CH2 domain that alter binding of the Fc by Fey receptors include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcyRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2): 132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcyRIIIa) (Stavenhagen, et al. 2007, Cancer Res 67(18):8882-90);F243L / R292P / Y300L / L235V / P396L (increased affinity for FcyRIIIa) (Nordstrom JL, et al., 2011,Breast Cancer Res, 13(6): R123); F243L (increased affinity for FcyRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcyRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcyRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005- 10), and S239D / S267E and S267E / L328F (increased affinity for FcyRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter binding of the Fc by FcyRIIb are described in International Publication No. WO 2021 / 232162. Additional modifications that affect Fc binding to Fey receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 379, Oct 2012, page 283).

[0141] Various publications describe strategies that have been used to engineer antibodies to produce “knock-out” variants (see, for example, Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, “Antibody Fc engineering for optimal antibody performance” In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include reduction of effector function through modification of glycosylation, use of IgG2 / IgG4 scaffolds, or the introduction of mutations in the hinge or CH2 domain of the Fc (see also, U. S. Patent Publication No. 2011 / 0212087, International Publication No. WO 2006 / 105338, U. S. Patent Publication No. 2012 / 0225058, U. S. Patent Publication No. 2012 / 0251531 and Strop et al., 2012, J. Mol. Biol., 420: 204-219). Examples of mutations that may be introduced into the hinge or CH2 domain to produce a “knock-out” variant include, but are not limited to, the amino acid modifications L234A / L235A, and L234A / L235A / D265S and the mutations described in International Publication No. WO 2023 / 028715.

[0142] Effector function may also be increased or decreased by modifying glycosylation of an FC, as is known in the art. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine or histidine (i.e. N297A, Q, K or H) results in an aglycoslated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601). Conversely, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC, based on improved binding to FcyRIIIa (see, for example, Shields et al., 2002, J Biol Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low fucose antibodies may be produced, for example inknockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622); in the variant CHO cell line, Lee 13, that has a reduced ability to attach fucose to N297-linked carbohydrates (International Publication No. WO 03 / 035835), or in other cells that generate afucosylated antibodies (see, for example, Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002, ibid, and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). In addition, International Publication No. WO 2009 / 135181 describes the addition of fucose analogues to culture medium during antibody production to inhibit incorporation of fucose into the carbohydrate on the antibody. Other methods of producing antibodies with little or no fucose on the Fc glycosylation site (N297) are well known in the art. For example, the GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U. S. Patent No. 8,409,572).

[0143] Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, for example, International Publication No. WO 2003 / 011878, U. S. Patent No. 6,602,684 and US Patent Application Publication No. US 2005 / 0123546). Useful glycosylation variants also include those having at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved GDC function (see, for example, International Publication Nos. WO 1997 / 030087, WO 1998 / 58964 and WO 1999 / 22764).

[0144] Other amino acid mutations in the CH2 domain that may be useful include amino acid mutations that result in increased binding to the neonatal Fc receptor (FcRn). Increased binding to FcRn may improve the in vivo half-life of the antibody construct. Amino acid substitutions in the CH2 domain that enhance binding to FcRn include, for example, the amino acid substitutions M252Y / S254T / T256E (“YTE mutations”) in both chains of the CH2 domain. In certain embodiments, the anti-PTK7 antibody constructs may comprise YTE mutations.

[0145] In certain embodiments, the anti-PTK7 antibody constructs have the format of a full-size antibody (FSA) and comprise two antigen-binding domains operably linked to an IgG Fc region via an IgG hinge region, where the Fc region comprises a CH2 and CH3 domain. In someembodiments, the anti-PTK7 antibody constructs have the format of an IgG FSA, for example, an IgGl FSA. In some embodiments, the anti-PTK7 antibody construct is a FSA comprising a first heavy chain sequence (HC1), a second heavy chain sequence (HC2), a first light chain sequence (LC1) and a second light chain sequence (LC2). In some embodiments, the anti-PTK7 antibody construct is a monospecific FSA with a homodimeric Fc and comprises HC1, HC2, LC1 and LC2 sequences, where HC1 and HC2 have the same amino acid sequence, and LC1 and LC2 have the same amino acid sequence. In some embodiments, the anti-PTK7 antibody construct is a monospecific FSA with a heterodimeric Fc and comprises HC1, HC2, LC1 and LC2 sequences, where HC1 and HC2 have different amino acid sequences, and LC1 and LC2 have the same amino acid sequence. In some embodiments, the anti-PTK7 antibody construct is a bispecific or biparatopic FSA with a heterodimeric Fc and comprises HC1, HC2, LC1 and LC2 sequences, where HC1 and HC2 have different amino acid sequences, and LC1 and LC2 have different amino acid sequences.BIPARATOPIC ANTI-PTK7 ANTIBODY CONSTRUCTS

[0146] In certain embodiments, the anti-PTK7 anlibody constructs are biparatopic anli-P'l’K7 antibody constructs. The biparatopic anti-PTK7 antibody constructs of the present disclosure comprise two or more of the PTK7 antigen-binding domains described herein, where at least two of the PTK7 antigen-binding domains bind to different epitopes on PTK7. In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise two PTK7 antigen-binding domains, each of which binds to a different epitope on PTK7.

[0147] In certain embodiments, each of the PTK7 antigen-binding domains of the biparatopic anti-PTK7 antibody construct may be an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. For example, a Fab fragment, a Fab’ fragment, a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb) as described herein. The biparatopic anti-PTK7 anlibody constructs may optionally comprise a scaffold, such as one of the scaffolds described herein. In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise an Fc region as a scaffold and at least one of the PTK7 antigen-binding domains is operably linked to the Fc region. In certain embodiments, the Fc region is a heterodimeric Fc region comprising amino acid substitutions in the CH3 domain to promoteheterodimer formation over homodimer lonnalion. Examples of amino acid substitutions that may be made in the CH3 domain to promote heterodimer formation are described herein.

[0148] In certain embodiments, each of the PTK7 antigen-binding domains comprised by the biparatopic anti-PTK7 antibody construct is independently a Fab or an scFv. In some embodiments, the biparatopic anti-PTK7 antibody constructs comprise two PTK7 antigen-binding domains, each of which is independently a Fab or an scFv, and an Fc region, where at least one of the PTK7 antigen-binding domains is operably linked to the Fc region. In some embodiments, the biparatopic anti-PTK7 anlibody constructs comprise two PTK7 anligen-binding domains, each of which is independently a Fab or an scFv, and an Fc region, where both PTK7 antigen-binding domains are operably linked to the Fc region. In some embodiments, the biparatopic anti-PTK7 anlibody constructs comprise two PTK7 antigen-binding domains, each of which is a Fab, and an Fc region, where both PTK7 anligen-binding domains are operably linked to the Fc region. In certain embodiments, the Fc region is a heterodimeric Fc region comprising amino acid subslilulions in the CH3 domain to promote heterodimer formation over homodimer formation.

[0149] In certain embodiments, the biparatopic anti-PTK7 antibody constructs have the format of a full-size antibody (FSA) and comprise two PTK7 antigen-binding domains operably linked to an IgG Fc region via an IgG hinge region, where the Fc region comprises a CH2 and CH3 domain. In certain embodiments, the Fc region is a heterodimeric Fc region comprising amino acid subslilulions in the CH3 domain to promote heterodimer formation over homodimer formation.

[0150] In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise:(a) a first PTK7 anligen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the CDR sequences (ECDR1, ECDR2, ECDR3) of the VE domain having a sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36; and (b) a second PTK7 antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the CDR sequences (ECDR1, ECDR2, ECDR3) of the VE domain having a sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64,

[0151] where the CDR sequences are as defined by one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0152] In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise:(a) a first PTK7 antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain having a sequence as set forth in SEQ ID NO: 32, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain having a sequence as set forth in SEQ ID NO: 34; and(b) a second PTK7 antigen-binding domain comprising the CDR sequences (HCDR1, HCDR2, HCDR3) of the VH domain having a sequence as set forth in SEQ ID NO: 58, and the CDR sequences (LCDR1, LCDR2, LCDR3) of the VL domain having a sequence as set forth in SEQ ID NO: 62,

[0153] where the CDR sequences are as defined by one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0154] In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise:(a) a first PTK7 antigen-binding domain comprising an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16, 18, 88 or 101; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24, 26 or 89, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; and(b) a second PTK7 antigen-binding domain comprising an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences asset forth in any one of SEQ ID NOs: 38, 41, 44, 46, 48, 92, 93 or 94; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 39, 42, 49, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57.

[0155] In certain embodiments, the biparatopic anli-P'I’K? antibody constructs comprise:(a) a first PTK7 antigen-binding domain comprising an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; and(b) a second PTK7 antigen-binding domain comprising:(i) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 41, 44, 46 or 48; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 39, 42 or 49; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57; or(ii) an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57.

[0156] In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise:(a) a first PTK7 antigen-binding domain comprising an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; and(b) a second PTK7 antigen-binding domain comprising an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3having a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57.

[0157] In certain embodiments, the biparatopic anli-P'I’K? antibody construct comprises:(a) a first PTK7 antigen-binding domain comprising a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 2, and a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 3, and(b) a second PTK7 antigen-binding domain comprising a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 4, and a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 5,where both the first and second PTK7 antigen-binding domains retain the ability to bind human PTK7.

[0158] In certain embodiments, the biparatopic anti-PTK7 antibody construct comprises:(a) a first PTK7 antigen-binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36, and(b) a second PTK7 antigen-binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64,where both the first and the second PTK7 antigen-binding domains retain the ability to bind human PTK7.

[0159] In certain embodiments, the biparatopic anti-PTK7 antibody construct comprises:(a) a first PTK7 antigen-binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34, and(b) a second PTK7 antigen-binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58, and a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62,where both the first and the second PTK7 antigen-binding domains retain the ability to bind human PTK7.

[0160] In certain embodiments, the biparatopic anti-PTK7 antibody construct comprises:(a) a first PTK7 antigen-binding domain comprising a VH sequence comprising the sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33, and a VL sequence comprising the sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36, and (b) a second PTK7 antigen-binding domain comprising a VH sequence comprising the sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61, and a VL sequence comprising the sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64.

[0161] In certain embodiments, the biparatopic anti-PTK7 antibody construct comprises:(a) a first PTK7 antigen-binding domain comprising a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, and a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34, and(b) a second PTK7 antigen-binding domain comprising a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58, and a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62.

[0162] In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise two PTK7 antigen-binding domains in Fab format. In such embodiments, the CHI and CL domains of each of the PTK7 antigen-binding domains may comprise sets of inulalions to drive the correct pairing between the respective heavy and light chains of each PTK7 antigen-binding domain. Examples of sets of mutations that may be used in this context include those described in International Patent Publication Nos. WO 2014 / 082179, WO 2015 / 181805 and WO 2017 / 059551. In certain embodiments, the biparatopic anti-PTK7 antibody constructs comprise two PTK7 anligen-binding domains in Fab format and comprise sets of amino acid inulalions to drive the correct pairing between the respective heavy and light chains of each PTK7 antigen-binding domain. In some embodiments, the sets of amino acid mutations are Design 4478 or Design 2739 as shown in Table 4.Table 4: Sets of CH1 / CL Amino Acid Mutations to Promote Correct PairingDesign Chain1Mutations24478 CH1_HCA A 139 W_L 143E_K145T_Q 179ECL_LCA F116A_Q124R_L135V_T178RCH1_HCB Q179RCL_LCB Q 124E_L 135 W_T 178E_T 180E2739 CH1_HCA L143RCL_LCA Q124E_V133ECH1_HCB L143E_K145T_Q179ECL_LCB Q124R_T178R1HCA = heavy chain A; LCA = light chain A; HCB = heavy chain B; LCB = light chain B, where HCA pairs with LCA and HCB pairs with LCB.2Numbering of amino acid positions according to Kabat

[0163] In certain embodiments, the biparatopic anti-PTK7 antibody constructs of the present disclosure show higher internalization into PTK7-expressing cells than the corresponding monospecific antibodies and the reference anti-PTK7 antibody cofetuzumab.

[0164] Antibody internalization may be measured using art-known methods, for example, by a direct internalization method according to the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as the pHAb Dyes (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dyes (ThermoFisher Scientific Corporation, Waltham, MA) and Incucyte® Fabfluor-pH Antibody Labeling Reagent (Sartorius AG, Gottingen, Germany), and analysis techniques such as microscopy, FACS, high content imaging or other plate-based assays. An exemplary method using flow cytometry and fluorescent labelling of antibodies with a Fab fragment AF488 conjugate targeting anti-human IgG Fc is described in the Examples.

[0165] In certain embodiments, the biparatopic anti-PTK7 antibody construct is considered to demonstrate a higher internalization into PTK7-expressing cells than a reference antibody (corresponding monospecific antibody or cofetuzumab) when the amount of biparatopic anti-PTK7 antibody construct internalized into the PTK7-expressing cells is at least 1.2 times greater than the amount of reference antibody internalized into the same PTK7-expressing cells under the same test conditions. In certain embodiments, the biparatopic anti-PTK7 antibody construct is considered to demonstrate a higher internalization into PTK7-expressing cells than a reference antibody (corresponding monospecific antibody or cofetuzumab) when the amount of biparatopic anti-PTK7 antibody construct internalized into the PTK7-expressing cells is at least 1.5 times greater, 1.6 times greater, 1.7 times greater, 1.8 times greater, 1.9 times greater, or 2.0 times greater, than the amount of reference antibody internalized into the same PTK7-expressing cells under the same test conditions. In some embodiments, the amount of internalized antibody is determined in cells that express PTK7 at a high level. In some embodiments, the amount of internalized antibody is determined in HCC1569 cells or cells that express PTK7 at a similar level to HCC1569 cells. In some embodiments, the amount of internalized antibody is determined in cells that express PTK7 at a mid level. In some embodiments, the amount of internalized antibodyis determined in H446 cells cells or cells that express PTK7 at a similar level to H446 cells. In some embodiments, the amount of internalized antibody is determined after a 4-hour incubation period. In some embodiments, the amount of internalized antibody is determined after a 6-hour incubation period. In some embodiments, the amount of internalized antibody is determined after a 24-hour incubation period.PREPARATION OF ANTI-PTK7 ANTIBODY CONSTRUCTS

[0166] The anti-PTK7 antibody constructs described herein may be produced using standard recombinant methods known in the art (see, for example, U. S. Patent No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0167] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-PTK7 antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotide(s) encoding the anti-PTK7 antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and “Antibodies: A Laboratory Manual,” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As would be appreciated by one of skill in the art, the number of polynucleotides required for expression of the anti-PTK7 antibody construct will be dependent on the format of the construct, including whether or not the antibody construct comprises a scaffold. For example, when an anti-PTK7 antibody construct is in a monospecific mAb format with a homodimeric Fc, two polynucleotides each encoding one polypeptide chain will be required, whereas when an anti-PTK7 antibody construct is in a monospecific mAb format with a heterodimeric Fc, three polynucleotides each encoding one polypeptide chain will be required. Analogously, when an anti-PTK7 antibody construct is in a mAb format and is bispecific or biparatopic with a heterodimeric Fc, four polynucleotides each encoding one polypeptide chain will be required. When multiple polynucleotides are required, they may be incorporated into one vector or into more than one vector.

[0168] Generally, for expression, the polynucleotide or set of polynucleotides encoding the anti-PTK7 antibody construct is incorporated into an expression vector or vectors together with one ormore regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. One skilled in the art will appreciate that the choice of regulatory elements is dependent on the host cell selected for expression of the antibody construct and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The expression vector may optionally further contain heterologous nucleic acid sequences that facilitate expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal-affinity tags, histidine tags, avidin / streptavidin encoding sequences, glutathione-S-transferase (GST) encoding sequences and biotin encoding sequences. The expression vector may be an extrachromosomal vector or an integrating vector.

[0169] Suitable host cells for cloning or expression of the anti-PTK7 antibody constructs include various prokaryotic or eukaryotic cells as known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells and yeast cells (such as Saccharomyces or Pichia cells). Prokaryotic host cells include, for example, E. coli, A. salmonicida or B. subtilis cells.

[0170] In certain embodiments, the anti-PTK7 antibody construct may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, as described for example in U. S. Patent Nos. 5,648,237; 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, B. K. C. Lo, ed., Humana Press, Totowa, N. J., 2003.

[0171] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, in particular fungi and yeast strains whose glycosylation pathways have been “humanized” resulting in the production of an antibody construct with a partially or fully human glycosylation pattern (see, for example, Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0172] Suitable host cells for the expression of glycosylated anti-PTK7 antibody constructs are usually eukaryotic cells. For example, U. S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension may beparticularly useful for expression of antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, for example, Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse sertoli TM4 cells (see, for example, Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumour (MMT 060562), TRI cells (see, for example, Mather et al., 1982, Annals N. Y. Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR“CHO cells, see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NSO and Sp2 / 0). Exemplary mammalian host cell lines suitable for production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (B. K. C. Lo, ed., Humana Press, Totowa, N. J., 2003).

[0173] In certain embodiments, the host cell used for expression of the anti-PTK7 antibody construct may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NSO or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.

[0174] The host cells comprising the expression vector(s) encoding the anti-PTK7 antibody construct may be cultured using routine methods to produce the anti-PTK7 antibody construct. Alternatively, in some embodiments, host cells comprising the expression vector(s) encoding the anti-PTK7 antibody construct may be used therapeutically or prophylactically to deliver the anti-PTK7 antibody construct to a subject, or polynucleotides or expression vectors may be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject.

[0175] Typically, the anti-PTK7 antibody constructs are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rdEd., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse-phase, carried out atatmospheric pressure or at high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins may be used for purification of certain antibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Likewise, the bacterial protein L binds to the Fab region of some antibodies. Purification may also be enabled by a particular fusion partner. For example, antibodies may be purified using glutathione resin if a GST fusion is employed, Ni+2affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flag-tag is used. The degree of purification necessary will vary depending on the use of the anti-PTK7 antibody constructs. In some instances, no purification may be necessary.

[0176] In certain embodiments, the anti-PTK7 antibody constructs are substantially pure. The term “substantially pure” (or “substantially purified”) when used in reference to an anti-PTK7 antibody construct described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, such as a native cell, or a host cell in the case of recombinantly produced construct. In certain embodiments, an anti-PTK7 antibody construct that is substantially pure is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.

[0177] Certain embodiments of the present disclosure relate to a method of making an anti-PTK7 antibody construct comprising culturing a host cell into which one or more polynucleotides encoding the anti-PTK7 antibody construct, or one or more expression vectors encoding the anti-PTK7 antibody construct, have been introduced, under conditions suitable for expression of the anti-PTK7 antibody construct, and optionally recovering the anti-PTK7 antibody construct from the host cell (or from host cell culture medium).Post- Translational Modifications

[0178] In certain embodiments, the anti-PTK7 antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications mayoccur in vivo, or they be conducted in vitro after isolation of the anti-PTK7 antibody construct from the host cell.

[0179] Post-translational modifications include various modifications as are known in the art (see, for example, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. N. Y. Acad. Sci., 663:48-62). In those embodiments in which the anti-PTK7 antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

[0180] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4.

[0181] Other examples of post-translational modifications include, for example, addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, and addition or deletion of an N-terminal methionine residue resulting from prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label, such as an enzymatic, fluorescent, luminescent, isotopic or affinity label to allow for detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. Examples of luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon and fluorine.

[0182] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.POLYNUCLEOTIDES, VECTORS AND HOST CELLS

[0183] Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding an anti-PTK7 antibody construct described herein. A polynucleotide in this context may encode all or part (for example, a heavy chain or a light chain) of an anti-PTK7 antibody construct.

[0184] The terms “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0185] A polynucleotide that “encodes” a given polypeptide is a polynucleotide that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0186] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an anti-PTK7 antibody constructdescribed herein. The polynucleotide(s) may be comprised by a single vector or by more than one vector. In some embodiments, the polynucleotides may be comprised by a multicistronic vector.

[0187] Certain embodiments of the present disclosure relate to host cells comprising polynucleotide(s) encoding an anti-PTK7 antibody construct described herein or one or more vectors comprising the polynucleotide(s). In some embodiments, the host cell is a mammalian host cell.ANTIBODY-DRUG CONJUGATES

[0188] Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising an anti-PTK7 antibody construct as described herein conjugated to one or more drug moieties, such as cytotoxins or immune modulators.

[0189] Typically, in an ADC, the anti-PTK7 antibody construct is conjugated to a drug moiety via a linker, which may be a cleavable or non-cleavable linker. The anlibody construct may be conjugated to a single drug molecule, or it may be conjugated to multiple drug molecules. The number of drug molecules conjugated to a single anlibody construct is defined by the drug-to-antibody ratio (DAR). In certain embodiments, in the ADCs of the present disclosure, the DAR is in the range of from about 1 to about 12, or from about 2 to about 12, or from about 2 to about 8. When the ADC comprises multiple drug moieties (i.e. the DAR is > 2), the drug moieties may be the same or they may be different. In some embodiments, the ADCs of the present disclosure comprise an anti-PTK7 antibody construct conjugated to more than one drug moiety and the drug moieties are the same. In some embodiments, the ADCs of the present disclosure comprise an anti-PTK7 antibody construct conjugated to more than one drug moiety and the drug moieties are different.

[0190] In certain embodiments, the ADCs comprising an anti-PTK7 antibody construct have the general Formula (I):A-(L-(D)m)n (I)

[0191] where A is an anti-PTK7 antibody construct as described herein; L is a linker; D is a drug moiety; m is between 1 and 8, and n is between about 1 and about 12.

[0192] In certain embodiments in Formula (I), m is between 1 and 6. In some embodiments, m is 1 or 2. In some embodiments, n is between about 1 and about 8, for example, between about 2 and about 8, or between about 4 and about 8. In some embodiments, m is 1 or 2 and n is between about 4 and about 8.Drug Moiety

[0193] Various compounds known to be useful as cytotoxic or immunomodulatory ADC payloads may be employed as the drug moiety in the ADCs comprising the anti-PTK7 antibody constructs. Examples include, but are not limited to, maytansinoids and maytansinoid analogues, benzodiazepines and pyrrolobenzodiazepines (PBDs, including PBD dimers), duocarmycins such as CC-1065 and analogues thereof, calicheamicins and calicheamicin analogues, auristatins and auristatin analogues, hemiasterlins and hemiasterlin analogues, tubulysins and tubulysin analogues, amatoxins and amatoxin analogues, topoisomerase 1 inhibitors including camptothecins and camptothecin analogues, eribulin, TLR agonists (such as agonists of TLR7 and / or TLR8) and STING agonists.

[0194] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is an auristatin or auristatin analogue, a hemiasterlin or a hemiasterlin analogue, a camptothecin or camptothecin analogue. In some embodiments, the drug moiety comprised by the ADCs of the present disclosure may be an auristatin analogue, such as MMAE, MMAF or an auristatin analogue as described in International Publication No. WO 2016 / 041082. In some embodiments, the drug moiety comprised by the ADCs of the present disclosure may be a hemiasterlin analogue, for example, as described in International Publication No. WO 2014 / 144871. In some embodiments, the drug moiety comprised by the ADCs of the present disclosure may be a camptothecin analogue, such as exatecan, Dxd or a camptothecin analogue as described in International Publication No. WO 2022 / 246576.

[0195] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is a camptothecin analogue having Formula (IV):wherein:R9NHR4is I;Xaand Xbare each O, andR9is selected from: -H, -C1-C6hydroxyalkyl, -C1-C6aminoalkyl, -aminoaryl and -(C1-C6alkyl)-aminoaryl.

[0196] In certain embodiments, in camptothecin analogues of Formula (IV), R9is -C1-C6hydroxyalkyl.

[0197] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is a camptothecin analogue, Compound 1, having the following structure:Compound 1

[0198] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is Compound 1, where the drug moiety is attached to linker, L, as shown below:where * is the point of attachment to linker, L.Linker

[0199] Typically, in the ADCs of the present disclosure, the drug moiety is linked to the antibody construct by a linker. Linkers are bi functional or multi functional moieties capable of linking one or more drug molecules to the antibody construct. In some embodiments, the linker may be bifunctional (or monovalent) such that it links a single drug molecule to a single site on the antibody construct. In some embodiments, the linker may be multi functional (or polyvalent) such that it links more than one drug molecule to a single site on the antibody construct. Multifunctional linkers may also be used to link one drug molecule to more than one site on the antibody construct in some embodiments.

[0200] Attachment of a linker to an antibody construct can be accomplished in a variety of ways, such as through surface lysines, reductive-coupling to oxidized carbohydrates, or through cysteine residues liberated by reducing interchain disulfide linkages. Alternatively, attachment of a linker to an antibody construct may be achieved by modification of the antibody construct to include additional cysteine residues (see, for example, U. S. Patent Nos. 7,521,541; 8,455,622 and 9,000,130) or non-natural amino acids that provide reactive handles, such as selenomethionine, p-acetylphenylalanine, formylglycine or p-azidomethyl-L-phenylalanine to allow for site-specific conjugation (see, for example, Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman etal., 2014, Bioconj. Chem., 25:351-361). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodelling of the antibody glycans to allow for attachment of a linker by metal-free click chemistry (see, for example, European Patent No. EP 2911 699).

[0201] Linkers typically include a functional group capable of reacting with the target group or groups on the antigen binding construct and one or more functional groups capable of reacting with a target group on the drug moiety. Suitable functional groups are known in the art and include those described, for example, in Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press). Non-limiting examples of functional groups for reacting with free cysteines or thiols include maleimide, haloacetamide, haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this context are “selfstabilizing” maleimides such as those described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062. Non-limiting examples of functional groups for reacting with surface lysines and amines include activated esters (such as N-hydroxy succinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Traut’s reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA)). Other examples include the use of succinimido-l,l,3,3-tetra-methyluronium tetrafluoroborate (TSTU) or benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) to convert a carboxylic acid to an activated ester, which may then be reacted with an amine. Non-limiting examples of functional groups capable of reacting with an electrophilic group on the antibody construct or drug moiety (such as an aldehyde or ketone carbonyl group) include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.

[0202] In certain embodiments, a linker that includes a functional group that allows for bridging of two interchain cysteines on the antibody binding construct may be used, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem., 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm., 12:3986-3998), a dithioaryl(TCEP)pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802) or a dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).

[0203] A variety of linkers for linking drugs to antibodies are known in the art, including hydrazone-, disulfide- and peptide-based linkers. Linkers may be cleavable or non-cleavable. A cleavable linker is typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include linkers that are protease-sensitive, acid-sensitive or reduction-sensitive. Non-cleavable linkers by contrast, rely on the degradation of the antibodyin the cell, which typically results in the release of an amino acid-linker-drug moiety. In certain embodiments, the ADCs of Formula (I) comprise a cleavable linker.

[0204] An example of a cleavable linker that may be useful in certain embodiments is a peptide-containing linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. Examples include dipeptide-containing linkers, such as those comprising the dipeptides Val-Cit, Phe-Lys, Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Vai-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys or Met-(D)Lys; tripeptide-containing linkers such as those comprising the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe -Phe-Lys or (D)Ala-Phe-Lys, and tetrapeptide-containing linkers such as those comprising the tetrapeptides Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly or Ala-Leu-Ala-Leu.

[0205] Additional useful cleavable linkers include disulfide-containing linkers and linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimydyl-4-(2 -pyridyldithio) butanoate (SPDB) and N-succinimydyl-4-(2-pyridyldithio)-2-sulfo butanoate (sulfo-SPDB). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.

[0206] A further example of a cleavable linker is a linker comprising a P-glucuronide, which is cleavable by P-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf etal., 2002, Curr. Pharm. Des., 8:1391-1403).

[0207] Cleavable linkers may optionally further comprise one or more additional functionalities such as self-immolative / self-elimination groups, stretchers or hydrophilic moieties.

[0208] Self-immolative and self-elimination groups that find use in linkers include, for example, p-aminobenzyl (PAB) and p-aminobenzyloxycarbonyl (PABC) groups, methylated ethylenediamine (MED) and hemi-aminal groups. Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PABC or PABE group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U. S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology, 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem., 55:5867-5877). Self-immolative / self-elimination groups, alone or in combination, are often included in peptide -based linkers but may also be included in other types of linkers. In some embodiments, the linker may include one or more self-immolative / self-elimination groups, for example, a PABC group, a PABE group, or a combination of a PABC or PABE group and an MED.

[0209] Various stretchers that find use in linkers for ADCs are known in the art and include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers also function as hydrophilic moieties and may be particularly useful with hydrophobic drugs, although their use in linkers with other drugs is also contemplated in some embodiments.

[0210] In certain embodiments, a stretcher may have one of the following structures:— (CH2)t— C—. — (CH2CH2O)U— C—. — (CH2)t— (CH2CH2O)U— C—.5 5 5O 0— (CH2CH2O)U— (CH2)t— C —. 5 — (CH2)t— (CH2CH2O)U— (CH2)t— C — 5O R O O O— (CH2)t— C— N— (CH2)t— C —or— (CH2)t— C-N— (CH2CH2O)U— C—,wherein:R is H or Ci-Ce alkyl;t is an integer between 2 and 10, andu is an integer between 1 and 10.

[0211] In some embodiments, in ADCs of Formula (I), linker, L, is a cleavable linker having Formula II:#_z_[slrj.AA1_[AA2]_lxj_%(II)wherein:Z is a linking group that joins the linker to a target group on the antibody construct, A; Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site;X is a self-immolative group;q is 0 or 1;r is 1, 2 or 3;s is 0, 1 or 2;# is the point of attachment to the antibody construct, A, and% is the point of attachment to the drug moiety, D.

[0212] In some embodiments of the present disclosure, in ADCs of Formula (I), linker, L, is a cleavable linker having Formula (V):*-z s,4qAAiAA2]Y]T%(V)wherein:Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A;Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site;Y is -NH-CH2- or -NH-CH2-C(O)-;q is 0 or 1;r is 1, 2 or 3;v is 0 or 1;# is the point of attachment to the anti-Ly6E antibody construct, A, and% is the point of attachment to the drug moiety, D.

[0213] In some embodiments, in linkers of Formula (V), v is 1.

[0214] In some embodiments, in linkers of Formula (II) or Formula (V), q is 1.

[0215] In some embodiments, in linkers of Formula (II) or Formula (V):Zis O, where # is the point of attachment to A, and * is the point of attachment to the remainder of the linker.

[0216] In some embodiments, in linkers of Formula (II) or Formula (V), Str is selected from:O O O— (CH2)t— C—. 5 — (CH2CH2O)U— C—.5 — (CH2)t— (CH2CH2O)U— C—.5O 0— (CH2CH2O)U— (CH2)t— C —. 5 — (CH2)t— (CH2CH2O)U— (CH2)t— C — 5O R O O O— (CH2)t— C-N-(CH2)t— C—and— (CH2)t— C-N-(CH2CH2O)U-C—wherein:R is H or Ci-Ce alkyl;t is an integer between 2 and 10, andu is an integer between 1 and 10.

[0217] In some embodiments, in linkers of Formula (II) or Formula (V), Str is:o o— (CH2)t— c—or— (CH2CH2O)U— (CH2)t— c —

[0218] In some embodiments, in linkers of Formula (II) or Formula (V), AAi-[AA2]r is a dipeptide (z.e. r = 1). In some embodiments, in linkers of Formula (II) or Formula (V), AAi-[AA2]r has a sequence selected from: Ala-(D)Asp, Ala-Lys, Ala-Phe, Asn-Lys, Asn-(D)Lys, Asp-Val, His-Val, Ile-Cit, Ile-Pro, Ile-Val, Leu-Cit, MesLys-Pro, Met-Lys, Met-(D)Lys, NorVal-(D)Asp, Phe-Arg, Phe-Cit, Phe-Lys, PhenylGly-(D)Lys, Pro-(D)Lys, Trp-Cit, Vai-Ala, Val-(D)Asp, Val-Cit, Val-Gly, Val-Gln and Val-Lys.

[0219] In some embodiments, in linkers of Formula (II) or Formula (V), AAi-[AA2]r is a tripeptide (z.e. r = 2). In some embodiments, in linkers of Formula (II) or Formula (V), AAi-[AA2]r has a sequence selected from: Ala-Ala-Asn, Ala-Val-Cit, (D)Ala-Phe-Lys, Asp-Val- Ala, Asp-Val-Cit, Gly-Cit-Val, Lys-Val-Ala, Lys-Val-Cit, Met-Cit-Val, (D)Phe-Phe-Lys, and Asn-Pro-Val.

[0220] In some embodiments, in linkers of Formula (II) or Formula (V), AAi-[AA2]r is a tetrapeptide (z.e. r = 3). In some embodiments, in linkers of Formula (II) or Formula (V), AAi-[AA2]rhas a sequence selected from: Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly.

[0221] In some embodiments, ADCs of Formula (I) may comprise a disulfide-containing linker. In some embodiments, in ADCs of Formula (I), linker, L, is a cleavable linker having Formula (III):R R(HI)wherein:Z is a linking group that joins the linker to a target group on the antibody construct, A; Q is -(CH2)p- or -(CFLCFLOjq-, wherein p and q are each independently an integer between 1 and 10;each R is independently H or Ci-Ce alkyl;n is 1, 2 or 3;# is the point of attachment to the antibody construct, A, and% is the point of attachment to the drug moiety, D.

[0222] In some embodiments, ADCs of Formula (I) may comprise a [j-glucuronidc-conlaining linker.

[0223] Various non-cleavable linkers are known in the art for linking drugs to antibodies and may be useful in the ADCs of the present disclosure in certain embodiments. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the antibody, as well as a maleimido- or haloacetyl-based moiety for reaction with the drug, or vice versa. An example of such a non-cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l-carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs via a maleimide group which reacts with sulfhydryls (thiols, — SH), while the sulfo-NHS ester is reactive toward primary amines. Other non-liiniling examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate) (“long chain” SMCC or LC-SMCC), K-maleimidoundecanoic acid N-succinimidyl ester (KMUA), Y-maleimidobutyric acid N-succinimidyl ester (GMBS), E-maleimidocaproic acid N-hydroxy succinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-([3-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB) and N-(p-maleimidophenyl)isocyanate (PMPI). Other examples include those comprising a haloacetyl-based functional group such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).Preparation of ADCs

[0224] ADCs comprising an anti-PTK7 antibody construct as described herein may be prepared by one of several routes known in the art, employing standard organic chemistry reactions, conditions, and reagents (see, for example, Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press). For example, conjugation may be achieved by (1) reaction of a functional groupof an antibody construct with a bivalent linker reagent, to form antibody-linker intermediate A-L, via a covalent bond, followed by reaction of A-L with an acli vated drug moiety D; or (2) reaction of a functional group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction of D-L with a functional group of an antibody construct. Conjugabon methods (1) and (2) may be employed with a variety of antibody constructs, drug moieties, and linkers to prepare the ADCs described herein. Alternatively, the drug-linker may be synthesized as a unit from commercially available starting materials or intermediates and then conjugated to the anti-PTK7 antibody construct.

[0225] Suitable functional groups on drug moiety, D, for attachment of linker, L, in either of the above approaches include, but are not limited to, thiol groups, amine groups, carboxylic acid groups and hydroxyl groups. In some embodiments of the present disclosure, linker, L, is attached to drug moiety, D, via a hydroxyl or amine group on the compound.

[0226] Suitable functional groups on the anti-PTK7 anlibody construct, A, for attachment of linker, L, in either of the above approaches include sulfhydryl groups (for example, on the sidechain of cysteine residues), amino groups (for example, on the side-chain of lysine residues), carboxylic acid groups (for example, on the side-chains of aspartate or glutamate residues), and carbohydrate groups.

[0227] Certain embodiments of the present disclosure relate to methods of preparing an ADC of Formula (I) comprising conjugating drug-linker L-(D)mto the anti-PTK7 anlibody, A. Some embodiments relate to methods of preparing an ADC of Formula (I) comprising conjugating druglinker L-(D)mto native cysteine residues of the anti-PTK7 anlibody, A.

[0228] Various prepared linkers, linker components and drugs are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March’s Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., 2002, J. Org. Chem., 67:1866-1872; Frisch et al., 1997, Bioconj. Chem., 7:180-186; Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press), and Antibody-Drug Conjugates: Methods in Molecular Biology (Ducry (Ed.), 2013, Springer)). In addition, a number of pre-formed drug-linkers suitable for reaction with a selected antibody construct are also available commercially, for example, druglinkers comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from CreativeBioLabs (Shirley, NY). Various antibody drug conjugation services are also available commercially from companies such as Lonza Inc. (Allendale, NJ), AbzenaPLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramal Pharma Solutions (Grangemouth, UK).

[0229] The ADCs, once prepared, may be purified by standard techniques such as chromatography (for example, HPLC, size-exclusion, adsorption, ion exchange and / or affinity capture), dialysis and / or langenlial flow filtration. The average DAR of the prepared ADC may optionally be determined by standard techniques such as UV / VIS spectroscopic analysis, ELISA- based techniques, chromatography techniques such as hydrophobic interaction chromatography (HIC), UV-MALDI mass spectrometry (MS) or MALDI-TOF MS. In addition, distribution of drug-linked forms (for example, the fraction of the anti-PTK7 antibody construct containing zero, one, two, three, etc. conjugated drug moieties) optionally may be analyzed, for example by MS (with or without an accompanying chromatographic separation step), hydrophobic interaction chromatography, reverse-phase HPLC or iso-electric focusing gel electrophoresis (IEF) (see, for example, Wakankar et al., 2011, mAbs, 3:161-172).

[0230] In certain embodiments, the drug-linker comprised by the ADCs of Formula (I) has the structure of any one of DL-1, DL-2 or DL-3 shown below:o HH0, N..N-So(DL-2)(DL-3).

[0231] In certain embodiments, the ADCs of Formula (I) comprise the drug-linker DL-1, where the drug-linker is conjugated to anti-PTK7 antibody construct, A, via the maleimide group as shown below:where ** is the point of conjugation to the anti-PTK7 antibody construct, A.

[0232] In certain embodiments, the ADCs of Formula (I) have the following structure:wherein:A is an anti-PTK7 antibody construct as described herein;L is a linker, andn is between about 2 and about 8, for example, between about 4 and about 8.

[0233] In certain embodiments, the ADCs of Formula (I) have the following structure:wherein n is between about 4 and about 8.METHODS OF USE

[0234] Certain aspects of the present disclosure relate to the therapeutic or diagnostic use of the anti-PTK7 antibody constructs and ADCs described herein. PTK7 is upregulated in a variety of cancers and certain embodiments of the present disclosure thus relate to the methods of using the anli-P'l’K7 antibody constructs and ADCs in the treatment or diagnosis of a PTK7-expressing cancer.

[0235] Certain embodiments relate to methods of inhibiting the growth of PTK7-expressing tumor cells comprising contacting the cells with an anti-PTK7 antibody construct or ADC described herein. The cells may be in vitro or in vivo. In certain embodiments, the anti-PTK7 antibody constructs and ADCs may be used in methods of treating a PTK7-expressing cancer or tumor in a subject.

[0236] PTK7-expressing cancers are typically solid tumors. Examples include, but are not limited to, ovarian cancer, breast cancer (including triple negative breast cancer (TNBC)), lung cancers (such as non-small cell lung cancer (NSCLC)), colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, gastric cancer and cholangiocarcinoma. Certain embodiments of the present disclosure relate to methods of irealing a PTK7-expressing cancer with an anti-PTK7 antibody construct or ADC as described herein, where the cancer is ovarian cancer, breast cancer (including triple negative breast cancer (TNBC)), lung cancer (such as non-small cell lung cancer (NSCLC)), colorectal cancer, esophageal cancer, head and neck cancer, cervical cancer, gastric cancer or cholangiocarcinoma.

[0237] Treatment of a PTK7-expressing cancer may result in one or more of: alleviation of symptoms, shrinking the size of the tumor, inhibiting growth of the tumor, diminishing one or more direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, improving survival, increasing progression-free survival, remission and / or improving prognosis.

[0238] In certain embodiments, when used in the treatment of cancer, the anti-PTK7 antibody constructs or ADCs may be administered systemically to the subject to be treated, for example, by bolus injection or continuous infusion into the subject’s bloodstream. In certain embodiments, when used in the treatment of cancer, the anti-PTK7 anlibody constructs or ADCs may be administered to the subject locally at the site to be treated.

[0239] It is contemplated that the anti-PTK7 anlibody constructs or ADCs may be used alone or in combi nalion with one or more known chemotherapeutic or immunotherapeutic agents typically used in the treatment of cancer. Combinations of the anti-PTK7 antibody constructs or ADCs with standard chemotherapeutics or iininunolhcrapculics may act to improve the efficacy of the chemotherapeutic or immunotherapeutic and, therefore, may improve standard cancer therapies.This application can be important in the treatment of drug-resistant cancers which are not responsive to standard treatment. When used in conjunction with one or more known chemotherapeutic or immunotherapeutic agents, the anti-PTK7 antibody construct or ADC may be administered prior to, or after, administration of the chemotherapeutic or immunotherapeutic agents, or they may be administered concomitantly.

[0240] The dosage of the the anti-PTK7 antibody construct or ADC to be administered is not subject to defined limits, but it will a therapeutically effective amount. A “therapeutically effective amount” refers to that amount of an anti-PTK7 antibody construct or ADC described herein which, when administered to a subject, is sufficient to effect a treatment of the particular indication. A therapeutically effective amount of anti-PTK7 antibody construct or ADC in respect of cancer treatment may, for example, have one or more of the following effects: reduce the number of cancer cells, reduce the tumor size, inhibit cancer cell infiltration into peripheral organs, inhibit tumor metastasis, inhibit tumor growth; increase survival time and / or relieve to some extent one or more of the symptoms associated with the cancer. For cancer therapy, efficacy may alternatively be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0241] Certain embodiments relate to methods of detecting the presence of PTK7 in a biological sample, such as a sample comprising cells or tissue, using an anti-PTK7 antibody construct described herein. In some embodiments, the biological sample may have been taken from a patient, for example, a patient known or suspected to have a cancer. Some embodiments relate to methods of detecting the presence of PTK7 in a biological sample that comprise contacting the sample with an anti-PTK7 antibody construct described herein.

[0242] Certain embodiments relate to methods of diagnosing a disorder associated with increased expression of PTK7, such as a cancer, using an anti-PTK7 antibody construct described herein. The method of diagnosis may be an in vivo method in which the anti-PTK7 antibody construct is administered to the subject, or it may be an in vitro method in which a sample taken from the subject is contacted with the anti-PTK7 antibody construct. For in vivo methods, administration may be systemic or local.

[0243] In methods of detecting the presence of PTK7 or diagnosing a disorder associated with increased expression of PTK7, the anti-PTK7 antibody construct may be labelled with a detectable label, such as a fluorescent, luminescent, chromophoric, chemiluminescent, radioactive or enzymatic label, as is known in the art.PHARMACEUTICAL COMPOSITIONS

[0244] For therapeutic use, the anti-PTK7 antibody constructs and ADCs may be provided in the form of pharmaceutical compositions comprising the anti-PTK7 antibody construct or ADC and a pharmaceutically acceptable carrier or diluent. The compositions may be prepared by known procedures using well-known and readily available ingredients.

[0245] Pharmaceutical compositions may be formulated for administration to a subject by, for example, parenteral, oral (including, for example, buccal or sublingual), topical, rectal or vaginal routes, or by inhalation or spray. “Parenteral” administration may be subcutaneous injection, or intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal or intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to the subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution. Pharmaceutical compositions may be provided as unit dosage formulations.

[0246] In certain embodiments, pharmaceutical compositions comprising the anti-PTK7 antibody constructs or ADCs may be formulated for parenteral administration by infusion or in a unit dosage injectable form, for example, as lyophilized formulations or aqueous solutions.

[0247] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such asserum albumin or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0248] In certain embodiments, pharmaceutical compositions comprising the anti-PTK7 antibody constructs or ADCs may be in the form of a sterile injectable aqueous or oleaginous solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents that are known in the art. The sterile injectable solution or suspension may comprise the anti-PTK7 antibody construct or ADC in a non-toxic parentally acceptable diluent or solvent. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer’s solution, bacteriostatic water for injection (BWFI), phosphate-buffered saline, dextrose solution or isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, various bland fixed oils may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anesthetics, preservatives and / or buffering agents may also be included in the injectable solution or suspension.

[0249] In certain embodiments, pharmaceutical compositions comprising the anti-PTK7 antibody constructs or ADCs may be formulated for intravenous administration to a subject, for example a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and / or a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, anampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0250] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in “Remington: The Science and Practice of Pharmacy” (formerly “Remingtons Pharmaceutical Sciences”) Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).PHARMACEUTICAL KITS

[0251] Certain embodiments relate to pharmaceutical kits comprising an anti-PTK7 antibody construct or ADC as described herein.

[0252] The kit typically will comprise a container holding the anti-PTK7 antibody construct or ADC and a label and / or package insert on or associated with the container. The label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products. The label or package insert may further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle.

[0253] In addition to the container holding the anti-PTK7 antibody construct or ADC, the kit may optionally comprise one or more additional containers comprising other components of the kit. For example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution or dextrose solution), other buffers or diluents.

[0254] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. If appropriate, one or more components of the kit may be lyophilized or provided in a dryform, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).

[0255] The kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.CERTAIN EMBODIMENTS

[0256] The present disclosure also encompasses the following Embodiments 1-91.

[0257] Embodiment 1: An antibody construct comprising: a first antigen-binding domain that binds to human PTK7 and comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36, and a second antigen-binding domain that binds to human PTK7 and comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64, wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0258] Embodiment 2: The anlibody construct according to Embodiment 1, wherein the first antigen-binding domain comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 32, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 34.

[0259] Embodiment 3: The antibody construct according to Embodiment 1 or Embodiment 2, wherein the second antigen-binding domain comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 58, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 62.

[0260] Embodiment 4: The anlibody construct according to Embodiment 1, wherein the first antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 comprisinga sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16, 18, 88 or 101; an HCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24, 26 or 89, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 22 or 27.

[0261] Embodiment 5: The antibody construct according to Embodiment 1 or Embodiment 4, wherein the second antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 41, 44, 46, 48, 92, 93 or 94; an HCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 39, 42, 49, 90 or 91; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57.

[0262] Embodiment 6: The antibody construct according to Embodiment 1, wherein: (a) the first antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; and (b) the second antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 comprising asequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57.

[0263] Embodiment 7: The antibody construct according to Embodiment 1, wherein: (a) the first antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 10; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 11; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 12; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 23; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 24, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 22; and (b) the second antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 40; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 92; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 42; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 53; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 54, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 52.

[0264] Embodiment 8: The antibody construct according to any one of Embodiments 1 to 7, wherein the first antigen-binding domain comprises a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 2.

[0265] Embodiment 9: The antibody construct according to any one of Embodiments 1 to 8, wherein the first antigen-binding domain comprises a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 3.

[0266] Embodiment 10: The antibody construct according to any one of Embodiments 1 to 9, wherein the second antigen-binding domain comprises a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 4.

[0267] Embodiment 11: The antibody construct according to any one of Embodiments 1 to 10, wherein the second antigen-binding domain comprises a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 5.

[0268] Embodiment 12: The antibody construct according to any one of Embodiments 1 to 11, wherein the first antigen-binding domain comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33.

[0269] Embodiment 13: The antibody construct according to any one of Embodiments 1 to 12, wherein the first antigen-binding domain comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36.

[0270] Embodiment 14: The antibody construct according to any one of Embodiments 1 to 13, wherein the second antigen-binding domain comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61.

[0271] Embodiment 15: The antibody construct according to any one of Embodiments 1 to 14, wherein the second antigen-binding domain comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64.

[0272] Embodiment 16: The antibody construct according to Embodiment 1, wherein the first antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32.

[0273] Embodiment 17: The antibody construct according to Embodiment 1 or Embodiment 16, wherein the first antigen-binding domain comprises a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34.

[0274] Embodiment 18: The antibody construct according to any one of Embodiments 1, 16 and 17, wherein the second antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58.

[0275] Embodiment 19: The antibody construct according to any one of Embodiments 1 and 16 to 18, wherein the second antigen-binding domain comprises a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62.

[0276] Embodiment 20: The antibody construct according to Embodiment 1, wherein: (a) the first antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 32, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 34, and (b) the second antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 58, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 62.

[0277] Embodiment 21: The antibody construct according to any one of Embodiments 1 to 20, wherein the first and second antigen-binding domains are each independently a Fab or an scFv.

[0278] Embodiment 22: The antibody construct according to any one of Embodiments 1 to 21, wherein the first and second antigen-binding domains are each a Fab comprising a CHI domain and a CL domain.

[0279] Embodiment 23: The antibody construct according to Embodiment 22, wherein the CHI and CL domains of each Fab comprise sets of amino acid mutations to drive correct pairing between the CH1 domain and CL domain of the first antigen-binding domain and between the CH1 domain and the CL domain of the second antigen-binding domain.

[0280] Embodiment 24: The antibody construct according to Embodiment 23, wherein: (a) the CHI domain and CL domain of one of the first or second antigen-binding domains comprise the set of amino acid mutations: CH1: A139W_L143E_K145T_Q179E and CL: Fl 16A_Q124R_L135V_T178R, and the CHI domain and CL domain of the other antigen-binding domain comprise the set of amino acid mutations: CH1: Q179R and CL: Q124E_L135W_T178E_T180E, or

[0281] (b) the CHI domain and CL domain of one of the first or second antigen-binding domains comprise the set of amino acid mutations: CHI: L143R and CL: Q124E_V133E, and the CHI domain and CL domain of the other antigen-binding domain comprise the set of amino acid mutations: CH1: L143E_K145T_Q179E and CL: Q124R_T178R.

[0282] Embodiment 25: The antibody construct according to any one of Embodiment 1 to 24 further comprising a scaffold, wherein the first and second antigen-binding domains are operably linked to the scaffold.

[0283] Embodiment 26: The antibody construct according to Embodiment 25, wherein the scaffold comprises an IgG Fc region.

[0284] Embodiment 27: The antibody construct according to Embodiment 26, wherein the IgG Fc region is an IgGl Fc region.

[0285] Embodiment 28: The antibody construct according to Embodiment 26 or Embodiment 27, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein the Fc polypeptides comprise one or more amino acid substitutions to promote formation of the heterodimeric Fc region.

[0286] Embodiment 29: The antibody construct according to Embodiment 28, wherein the amino acid substitutions comprised by the Fc polypeptides are: (a) the amino acid substitutions L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T366L_K392M_T394W in the other Fc polypeptide, or (b) the amino acid substitutions L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T366L_K392L_T394W in the other Fc polypeptide, or (c) the amino acid substitutions T350V_L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392L_T394W in the other Fc polypeptide, or (d) the amino acid substitutions T350V_L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392M_T394W in the other Fc polypeptide, or (e) the amino acid substitutions T350V_L351Y_S400E_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_N390R_K392M_T394W in the other Fc polypeptide, wherein the numbering of amino acids is according to the EU index.

[0287] Embodiment 30: An antibody construct comprising: a first antigen-binding domain that binds to human PTK7 and comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 73, and a light chain comprising the sequence as set forth in SEQ ID NO: 75, and a second antigen-binding domain that binds to human PTK7 and comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 74, and a light chain comprising the sequence as set forth in SEQ ID NO: 76.

[0288] Embodiment 31: An antibody construct comprising an antigen-binding domain that binds to human PTK7, wherein the antigen-binding domain comprises the CDR (HCDR1, HCDR2, HCDR3) sequences of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the CDR (LCDR1, LCDR2, LCDR3) sequences of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36, and wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0289] Embodiment 32: The antibody construct according to Embodiment 31, wherein the antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27.

[0290] Embodiment 33: The antibody construct according to Embodiment 31, wherein the antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 10; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 11; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 12; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 23; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 24, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 22.

[0291] Embodiment 34: The antibody construct according to any one of Embodiments 31 to 33, wherein the antigen-binding domain comprises a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 2.

[0292] Embodiment 35: The antibody construct according to any one of Embodiments 31 to 34, wherein the antigen-binding domain comprises a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 3.

[0293] Embodiment 36: The antibody construct according to any one of Embodiments 31 to 35, wherein the antigen-binding domain comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33.

[0294] Embodiment 37: The antibody construct according to any one of Embodiments 31 to 36, wherein the antigen-binding domain comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36.

[0295] Embodiment 38: The antibody construct according to any one of Embodiments 31 to 37, wherein the antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32.

[0296] Embodiment 39: The antibody construct according to any one of Embodiments 31 to 38, wherein the antigen-binding domain comprises a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34.

[0297] Embodiment 40: The antibody construct according to any one of Embodiments 31 to 39, wherein the antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 32, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 34.

[0298] Embodiment 41: An antibody construct comprising an antigen-binding domain that binds to human PTK7, wherein the antigen-binding domain comprises the CDR (HCDR1, HCDR2, HCDR3) sequences of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the CDR (LCDR1, LCDR2, LCDR3) sequences of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64, and wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

[0299] Embodiment 42: The antibody construct according to Embodiment 41, wherein the antigen-binding domain comprises an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 52 or 57.

[0300] Embodiment 43: The antibody construct according to Embodiment 41, wherein the antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 40; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 92; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 42; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 53; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 54, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 52.

[0301] Embodiment 44: The antibody construct according to any one of Embodiments 41 to 43, wherein the antigen-binding domain comprises a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 4.

[0302] Embodiment 45: The antibody construct according to any one of Embodiments 41 to 44, wherein the antigen-binding domain that comprises a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 5.

[0303] Embodiment 46: The antibody construct according to any one of Embodiments 41 to 45, wherein the antigen-binding domain comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61.

[0304] Embodiment 47: The antibody construct according to any one of Embodiments 41 to 46, wherein the antigen-binding domain comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64.

[0305] Embodiment 48: The antibody construct according to any one of Embodiments 41 to 47, wherein the antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58.

[0306] Embodiment 49: The antibody construct according to any one of Embodiments 41 to 48, wherein the antigen-binding domain comprises a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62.

[0307] Embodiment 50: The antibody construct according to any one of Embodiments 41 to 49, wherein the antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 58, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 62.

[0308] Embodiment 51: The antibody construct according to any one of Embodiments 31 to 50, wherein the antigen-binding domain is a Fab or an scFv.

[0309] Embodiment 52: The antibody construct according to any one of Embodiments 31 to 51, further comprising a scaffold, wherein the antigen-binding domain is operably linked to the scaffold.

[0310] Embodiment 53: The antibody construct according to Embodiment 52, wherein the scaffold comprises an IgG Fc region.

[0311] Embodiment 54: The antibody construct according to any one of Embodiments 31 to 53, further comprising a second antigen-binding domain.

[0312] Embodiment 55: The antibody construct according to Embodiment 54, wherein the second antigen-binding domain binds to PTK7.

[0313] Embodiment 56: A polynucleotide or set of polynucleotides encoding the antibody construct according to any one of Embodiments 1 to 55.

[0314] Embodiment 57: An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to Embodiment 56.

[0315] Embodiment 58: A host cell comprising the polynucleotide or set of polynucleotides according to Embodiment 56 or the expression vector or set of expression vectors according to Embodiment 57.

[0316] Embodiment 59: An antibody-drug conjugate comprising the antibody construct according to any one of Embodiments 1 to 55 conjugated to one or more drug moieties.

[0317] Embodiment 60: The antibody-drug conjugate according to Embodiment 59, wherein the antibody conjugate is conjugated to between 1 and about 8 drug moieties.

[0318] Embodiment 61: An antibody-drug conjugate having general Formula (I): A-(L-(D)m)n (I), wherein: A is the antibody construct according to any one of Embodiments 1 to 55; L is a linker; D is a drug moiety; m is between 1 and about 8, and n is 1 and about 12.

[0319] Embodiment 62: The antibody-drug conjugate according to Embodiment 61, wherein m is 1 or 2.

[0320] Embodiment 63: The antibody-drug conjugate according to Embodiment 61 or Embodiment 62, wherein n is between about 2 and about 8.

[0321] Embodiment 64: The antibody-drug conjugate according to Embodiment 61 or Embodiment 62, wherein n is between about 4 and about 8.

[0322] Embodiment 65: The antibody-drug conjugate according to any one of Embodiments 59 to 64, wherein the drug moiety is a maytansinoid, maytansinoid analogue, benzodiazepine, pyrrolobenzodiazepine, duocarmycin, calicheamicin, calicheamicin analogue, auristatin, auristatin analogue, hemiasterlin, hemiasterlin analogue, tubulysin, tubulysin analogue, amatoxin, amatoxin analogue, camptothecin, camptothecin analogue, eribulin, TER agonist or STING agonist.

[0323] Embodiment 66: The antibody-drug conjugate according to any one of Embodiments 59 to 64, wherein the drug moiety is a camptothecin analogue or an auristatin analogue.

[0324] Embodiment 67: The antibody-drug conjugate according to any one of Embodiments 61 to 64, wherein the drug moiety is:

[00325]

[0326] Embodiment 68: The antibody-drug conjugate according to any one of Embodiments 61 to 64 and 67, wherein linker, L, has:

[0327] (a) Formula (II):'-z st,AAi-tAA2-lx]%

[0328] <“) wherein: Z is a linking group that joins the linker to a target group on the antibody construct, A; Str is a stretcher; AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site; X is a self-immolative group; q is 0 or 1; r is 1, 2 or 3; s is 0, 1 or 2; # is the point of attachment to the antibody construct, A, and % is the point of attachment to the drug moiety, D,

[0329] or (b) Formula (V):

[0330] '-z s,r-kAA<AA^Y17%(V), wherein: Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A; Str is a stretcher; AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site; Y is -NH-CH2- or -NH-CH2-C(O)-; q is 0 or 1; r is 1, 2 or 3; v is 0 or 1; # is the point of attachment to the anti-Ly6E antibody construct, A, and % is the point of attachment to the drug moiety, D.

[0331] Embodiment 69: The antibody-drug conjugate according to any one of Embodiments 61 to 64, wherein L-(D)mhas the structure:

[00332] where ** is the point of conjugation to the antibody construct, A.

[0333] Embodiment 70: An antibody-drug conjugate having the structure:

[00334]

[0335] wherein n is between about 4 and about 8, and wherein A is an antibody construct comprising: (a) the first antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 10; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 11; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 12; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 23; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 24, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 22; and (b) the second antigenbinding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 40; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 92; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 42; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 53; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 54, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 52.

[0336] Embodiment 71: The antibody-drug conjugate according to Embodiment 70, wherein: (a) the first antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 32, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 34, and (b) the second antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 58, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 62.

[0337] Embodiment 72: The antibody-drug conjugate according to Embodiment 70 or Embodiment 71, wherein the first and second antigen-binding domains are each a Fab comprising a CHI domain and a CL domain.

[0338] Embodiment 73: The antibody-drug conjugate according to Embodiment 72, wherein the CHI and CL domains of each Fab comprise sets of amino acid mutations to drive correct pairing between the CH1 domain and CL domain of the first antigen-binding domain and between the CH1 domain and the CL domain of the second antigen-binding domain.

[0339] Embodiment 74: The antibody-drug conjugate according to claim 73, wherein:

[0340] (a) the CHI domain and CL domain of one of the first or second antigen-binding domains comprise the set of amino acid mutations: CH1: A139W_L143E_K145T_Q179E and CL: Fl 16A_Q124R_L135V_T178R, and the CHI domain and CL domain of the other antigen-bindingdomain comprise the set of amino acid mutations: CH1: Q179R and CL: Q124E_E135W_T178E_T180E, or

[0341] (b) the CHI domain and CL domain of one of the first or second antigen-binding domains comprise the set of amino acid mutations: CHI: L143R and CL: Q124E_V133E, and the CHI domain and CL domain of the other antigen-binding domain comprise the set of amino acid mutations: CH1: L143E_K145T_Q179E and CL: Q124R_T178R.

[0342] Embodiment 75: The antibody-drug conjugate according to any one of Embodiments 70 to 74 further comprising a scaffold, wherein the first and second antigen-binding domains are operably linked to the scaffold, and wherein the scaffold comprises an IgG Fc region.

[0343] Embodiment 76: The antibody-drug conjugate according to Embodiment 75, wherein the IgG Fc region is an IgGl Fc region.

[0344] Embodiment 77: The antibody construct according to Embodiment 75 or Embodiment 76, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein the Fc polypeptides comprise one or more amino acid substitutions to promote formation of the heterodimeric Fc region.

[0345] Embodiment 78: The antibody-drug conjugate according to Embodiment 77, wherein the amino acid substitutions comprised by the Fc polypeptides are: (a) the amino acid subslilulions L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T366L_K392M_T394W in the other Fc polypeptide, or (b) the amino acid substitutions L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T366L_K392L_T394W in the other Fc polypeptide, or (c) the amino acid substitutions T350V_L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392L_T394W in the other Fc polypeptide, or (d) the amino acid substitutions T350V_L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392M_T394W in the other Fc polypeptide, or (e) the amino acid substitutions T350V_L351Y_S400E_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_N390R_K392M_T394W in the other Fc polypeptide, wherein the numbering of amino acids is according to the EU index

[0346] Embodiment 79: The antibody-drug conjugate according to Embodiment 70, wherein the first antigen-binding domain comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 73, and a light chain comprising the sequence as set forth in SEQ ID NO: 75, and the second antigen-binding domain comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 74, and a light chain comprising the sequence as set forth in SEQ ID NO: 76.

[0347] Embodiment 80: A method of preparing the antibody-drug conjugate according to any one of Embodiments 61 to 69 comprising conjugating drug -linker L-(D)mto the antibody construct, A.

[0348] Embodiment 81: The method according to Embodiment 80, wherein drug-linker L-(D)mis conjugated to native cysteine residues of the antibody construct, A.

[0349] Embodiment 82: A method of preparing the antibody-drug conjugate according to any one of Embodiments 70 to 79 comprising conjugating a drug-linker having the structure:H H U N N. AN Y N N O'HJ H H HNto the antibody construct, A.

[0350] Embodiment 83: The method according to Embodiment 82, wherein the drug-linker is conjugated to native cysteine residues of the antibody construct, A.

[0351] Embodiment 84: A pharmaceutical composition comprising the antibody construct according to any one of Embodiments 1 to 55, and a pharmaceutically acceptable carrier or diluent.

[0352] Embodiment 85: A pharmaceutical composition comprising the antibody-drug conjugate according to any one of Embodiments 59 to 79, and a pharmaceutically acceptable carrier or diluent.

[0353] Embodiment 86: An antibody construct according to any one of Embodiments 1 to 55 for use in therapy.

[0354] Embodiment 87: The antibody construct for use according to Embodiment 86, wherein the therapy comprises treatment of cancer.

[0355] Embodiment 88: An antibody-drug conjugate according to any one of Embodiments 59 to 79 for use in therapy.

[0356] Embodiment 89: The antibody-drug conjugate for use according to Embodiment 88, wherein the therapy comprises treatment of cancer.

[0357] Embodiment 90: Use of an antibody construct according to any one of Embodiments 1 to 55 in the manufacture of a medicament for the treatment of cancer.

[0358] Embodiment 91: Use of an antibody-drug conjugate according to any one of Embodiments 59 to 79 in the manufacture of a medicament for the treatment of cancer.

[0359] Embodiment 92: A method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody construct according to any one of Embodiments 1 to 55.

[0360] Embodiment 93: A method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody-drug conjugate according to any one of Embodiments 59 to 79.

[0361] Embodiment 94: A method of treating a subject having a cancer comprising administering to the subject an effective amount of the antibody construct according to any one of Embodiments 1 to 55.

[0362] Embodiment 95: A method of treating a subject having a cancer comprising administering to the subject an effective amount of the antibody-drug conjugate according to any one of Embodiments 59 to 79.

[0363] The following Examples are provided for illustrative purposes and are not intended to limit the scope of the claimed invention in any way.EXAMPLES EXAMPLE 1: PREPARATION OF ANTI-PTK7 ANTIBODIES

[0364] Antibodies that specifically bind PTK7 were generated by immunizing mice as described below.

[0365] Briefly, ten CD1 mice were immunized by hydrodynamic tail vein injection with a pTT5 based expression plasmid (National Research Council of Canada) encoding human PTK7 (SEQ ID NO: 1, see Table 2) over 63 days, after which blood was drawn and spleens harvested.

[0366] Anti-human PTK7 antibody titers were determined by flow cytometry using streptavidin beads (Spherotech Inc., Greenoaks, IL) coated with recombinant huPTK7 antigen (R& D Systems, Minneapolis, MN; Cat No. 9799-TK). Terminal bleed sera mounted a significant response against human PTK7.

[0367] Immunized mice were sacrificed, and the spleens harvested and pooled. Splenocytes were used for B cell enrichment and sorted on a FACSAria™ cell sorter (Becton, Dickinson & Co., Franklin Lakes, NJ) into wells containing lysis buffer with a modified protocol based on the Selected Lymphocyte Antibody Method (SLAM) (Babcook et al., 1996, Proc Natl Acad Sci USA, 93(15):7843-7848).

[0368] Total RNA from wells containing a single B cell was used as template with SuperScript™ III (Thermo Fisher Scientific Corp., Waltham, MA) and oligo-dT20 (Integrated DNA Technologies, Inc., Coralville, IA) to transcribe cDNA from mRNA. Initial PCR of heavy and light chain antibody-coding sequences was performed using primers and methods modified from Babcook et al., 1996, Proc Natl Acad Sci USA, 93(15):7843-7848, von Boehmer et al., 2016, Nat Protoc., 11(10): 1908 and Peng et al., 2017, J. Mol. Biol, 429(19):2954-2973, with cDNA as the nucleic acid template. A subsequent PCR reaction was then performed on these unique sequences using V-segment family and J-segment family- specific primers and the resulting amplicons were cloned into pTT5-based expression plasmids (National Research Council of Canada). Uniqueheavy chain sequences and light chain sequences emerging from a single well sample were coexpressed in Expi293F cells (Thermo Fisher Scientific, Waltham, MA; Cat No. A14527). Heavy and light chain PCR amplicons were sequenced using next generation sequencing (NGS)-based Amplicon-EZ and analyzed for unique antibody-coding sequences.

[0369] Cell supernatants containing secreted antibodies were assessed for human and cynomolgus monkey (cyno) PTK7 specificity by binding on HEK293-6E cells transiently transfected with a pTT5 based plasmid encoding human or cyno PTK7. 13G11 and 14D08 anti-PTK7 antibodies were selected for further evaluation. Sequences of the mouse VH and VL domains for these two antibodies are provided in Table 1.1 below.Table 1.1: Mouse Vu and VL Sequences for Anti-PTK7 Antibodies 13G11 and 14D08 Antibody VH / VL Sequence SEQ ID NO VH EVQLQQSGPELVKPGASVKISCKASGYALSRSWMNW 2 VKQRPGKGLEWIGRIYSGDGDIHYNGKFKGKATLTA DKSSNTAHMQLSSLTSEDSAVYFCAWGSSPFDIWGQ13G11 GTLVTVSAVL DIVLTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQ 3 KLHESPRLLIKYASQSIPGIPSRFSGSGSGSDFTLSINSV EPEDVGVYYCQNGHSFPWTFGGGTKLEIK VH QVQLKESGPGLVAPSQSLSITCTVSGFSLSSYGVHWV 4 RQPPGKGLEWLVLIWSDGSTTYNSALKSRLTISKDNS KSQVFLEMNSLQADDTAMYYCARHNGYYAMDYWG14D08 QGTSVTVSSVL DIVLTQSPSYLAASPGETITINCRASKSISKYLAWYQE 5 KPGKTNKLLIYSGSTLQFGIPSRFSGSGSGTDFTLTISSL EPEDFAMYYCQQHNEYPLTFGAGTKLELK

[0370] Mouse -human chimeric IgGl / kappa antibodies based on 13G11 and 14D08 (v39093 and v39090, respecti vely) were generated as follows. Each mouse VH domain sequence was appended to the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 containing T350V_L351Y_F405A_Y407V mutations (chain A) in one heavy chain and T350V_T366L_K392L_T394W mutations (chain B) in the second heavy chain (EU numbering), providing mouse-human chimeric full heavy chain sequences. Each mouse VL domain sequencewas appended to the human kappa CL sequence of IGKC*01 to provide a mouse-human chimeric light chain sequence. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0371] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 6) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA.

[0372] Transfection Conditions: Antibody variants, comprising heavy chain A and heavy chain B and light chain, were transiently expressed in CHO 3E7 cells.

[0373] Briefly, CHO-3E7 cells were cultured in FreeStyle™ F17 expression medium (Gibco Cat No. A- 1383501) supplemented with 4 mM L-glutamine (HyClone / Cytivia, Cat No. SH30034.01) and 0.1% Pluronic F-68™ (Gibco, Cat No. 24040-032) at 37°C. A transfection mixture of lOOug DNA (50ug DNA coding for the antibody and 50ug of Salmon Sperm DNA, pTT22-AKTdd, GFP) was complexed with PEI MAX® 40,000 (PolyScience, Cat No. 24765-1) at a 1:4 (w / w) DNA / PEI ratio. Transfections were performed with DNA / PEI complexes in lOOmL cell culture volumes supplemented with IX Antibiotic / Antimycotic Soludon (HyClone, Cat No. SV30079.01) and incubated at 37°C for 24 hrs. This was followed by a temperature shift to 32°C for 6 days with 0.5 mM valproic acid and 1.0% (w / v) peptone. GFP measurements were taken 1 day post-transfection to determine transfection efficiency.

[0374] Productions were harvested 7 days post-transfection. Cells were centrifuged then filtered on a 0.22uM Millipore filter. Clarified supernatants were analyzed by small scale HPLC -Protein A for titer measurements.

[0375] Protein A Affinity Chromatography and Preparative Size Exclusion Chromatography (SEC): Clarified culture medium was loaded onto a MabSelect™ SuRe™ (Cytiva) Protein A column and washed with Dulbecco’s phosphate -buffered saline (DPBS) buffer (pH 7.0-7.2).Protein was eluted with lOOmM citrate buffer at pH 3.0 and neutralized with 10-15% (v / v) IM HEPES solution to yield a final pH of 6-7. Samples underwent preparative SEC (Superdex™ 200 Increase 10 / 300 GL, Cytiva) in DPBS mobile phase. Protein was quantified based on absorbance at 280nm (A280nm). Final yields were ~3.8 mg and -3.3 mg for v39090 and v39093, respectively.

[0376] Caliver: Purity of samples was assessed by electrophoresis under non-reducing (NR) and reducing (R) conditions using the High Throughput Protein Express assay and Caliper LabChip® GXII Touch HT system (Perkin Elmer, Waltham, MA). Procedures were carried out according to HT Protein Express LabChip® User Guide version 2 with the following modifications. Antibody samples, at either 2 pl or 5 pl (concentration range 5-2000 ng / pl), were added to separate wells in 96 well plates (BioRad, Hercules, CA; Cat. No. HSP9601) along with 7pl of HT Protein Express Sample Buffer (Perkin Elmer; Cat. No. 760518). Antibody samples were then denatured at 70°C for 15 mins. The LabChip® instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the HT Antibody Analysis 200 High Sensitivity assay setting.

[0377] NR Caliper results reflected predominantly single species corresponding to full-size antibody and R Caliper reflected intact heavy and light chains.

[0378] UPLC-SEC: Species homogeneity of the final antibody variant samples was assessed by UPLC-SEC. UPLC-SEC was performed using a Waters Acquity™ BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 pm particles) (Waters LTD, Mississauga, ON) set to 25°C and mounted on a Waters Acquity™ UPLC H-Class Bio system with a photodiode array (PDA) detector. The mobile phase was DPBS with 0.02% Tween 20 pH 7.4 and the flow rate was 0.4 mL / min. Total run time for each injection was 7 min with a total injection of lOug of protein sample. Elution was monitored by UV absorbance in the range 210-500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Waters Empower® 3 software employing the Apex Track™ and detect shoulders features.

[0379] UPLC-SEC profiles were reflective of relatively high species homogeneity. For v39090, -92% monomeric species purity was determined and for v39093, -97% monomeric species purity was determined.EXAMPLE 2: FUNCTIONAL CHARACTERIZATION OF CHIMERIC ANTI-PTK7 ANTIBODIES - COMPETITION BINDING (EPITOPE BINNING)

[0380] To characterize the binding of chimeric anti-PTK7 antibodies v39093 and v39090 to PTK7, competition binding (epitope binning) assays were carried out against a reference anti-PTK7 antibody based on cofetuzumab (vl9287). Binding was assessed by flow cytometry using NCI-H520 cells as described below.

[0381] vl9287 is identical to cofetuzumab but includes a heterodimeric Fc region comprising the mutations: T350V_L351Y_F405A_Y407V in chain A, and the mutations: T350V_T366L_K392L_T394W in chain B (EU numbering). The heavy chain and light chain sequences for vl9287 (cofetuzumab) are provided in Table 2.1.Table 2.1: Full-Length Heavy and Light Chain Amino Acid Sequences for vl9287 Name Amino Acid Sequence SEQ ID NOHeavy QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYAVHWVRQAPGKRLE 104 Chain A WIGVISTYNDYTYNNQDFKGRVTMTRDTSASTAYMELSRLRSEDTAV YYCARGNSYFYALDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGHeavy QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYAVHWVRQAPGKRLE 105 Chain B WIGVISTYNDYTYNNQDFKGRVTMTRDTSASTAYMELSRLRSEDTAV YYCARGNSYFYALDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIA VEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGLight EIVLTQSPATLSLSPGERATLSCRASESVDSYGKSFMHWYQQKPGQAP 106 Chain RLLIYRASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREName Amino Acid Sequence SEQ ID NO AKVQWKVDNAEQSGNSQESVTEQDSKDSTYSESSTETESKADYEKHK VYACEVTHQGESSPVTKSFNRGEC

[0382] Each of the anti-PTK7 detection antibodies, v39093 and v39090, vl9287 (cofetuzumab) and palivizumab (anti-RSV, v21995), was conjugated with AF647 fluorophores using Zenon™ Human IgG Labeling Kit (Thermo Fisher Scientific Inc., Waltham, MA; Cat. No. Z25408). NCI-H520 cells were cultured in 10cm3plates at 37°C / 5% CO2in ATCC-recommended growth media and detached using TrypEE™ Express (Invitrogen, Waltham, MA; Cat. No. 12604-013). Each well of a V-bottom 96-well plate was seeded with 50,000 cells and incubated with 100 pg / mL of unlabeled competitor anti-PTK7 antibody for an hour on ice. Post incubation, cells were stained with 1 pg / mL of AF647-conjugated anti-PTK7 detection antibodies for an hour on ice. Following staining and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1000 minimum events collected per well. The AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated for the live cell population and using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ). Percentage inhibition was calculated using the following formula: / Competitor mAb Geomean \% Competition = 100 — - - - - - x 100\ Irrelevant mAb Geomean )

[0383] The competition binding results for the chimeric anti-PTK7 antibodies v39093, and v39090 against cofetuzumab (vl9287) are shown in Table 2.2. v39093 competed against cofetuzumab (98%) and v39090 partially competed against cofetuzumab (73%). Cofetuzumab (vl9287) competed against itself as expected. No competition binding against negative control palivizumab (v21995) was observed, as expected.Table 2.2: Competition Binding - Percentage Inhibition vs. Irrelevant mAb (v21995)% Inhibition vs. Irrelevant mAbLabeled DetectionUnlabeled Competitor AntibodyAntibody No mAbV19287 v21995(cofetuzumab) (negative control)v39093 98 0 -1v39090 73 0 -3V1928799 0 0 (cofetuzumab)v219950 0 38*(negative control)**v21995-AF647 showed some non-specific bindingEXAMPLE 3: HUMANIZATION OF ANTI-PTK7 ANTIBODY 13G11 AND ASSESSMENT OF BINDING TO HUMAN AND CYNOMOLGUS PTK7

[0384] The chimeric anti -human PTK7 antibody, variant v39093, based on anti-PTK7 antibody 13G11 generated as described in Example 1, was humanized as described below. The CDR sequences of the chimeric antibody v39093, which are the same as those of the parental anti -PT K7 antibody 13G11, are provided in Table 3.1.Table 3.1: CDR Sequences of Chimeric Antibody v39093 (Anti-PTK7 Antibody 13G11) SequenceNumbering SEQ SEQ SEQ System Heavy Chain Heavy Chain CDR1 ID Heavy Chain CDR2 ID ID NO NO CDR3 NO IMGT GYALSRSW 7 IYSGDGDI 8 AWGSSPFDI 9Kabat RSWMN 10 RIYSGDGDIHYNGK 101 GSSPFDI 12 FKGChothia GYALSRS 13 YSGDGD 14 GSSPFDI 12 AbM GYALSRSWMN 15 RIYSGDGDIH 16 GSSPFDI 12 Contact SRSWMN 17 WIGRIYSGDGDIH 18 AWGSSPFD 19SEQ SEQ SEQLight Chain Light Chain CDR1 ID Light Chain CDR2 ID ID NO NO CDR3 NO IMGT QSISDY 20 YAS 21 QNGHSFPWT 22 Kabat RASQSISDYLH 23 YASQSIP 24 QNGHSFPWT 22 Chothia RASQSISDYLH 23 YASQSIP 24 QNGHSFPWT 22 AbM RASQSISDYLH 23 YASQSIP 24 QNGHSFPWT 22Contact SDYLHWY 25 LLIKYASQSI 89 QNGHSFPW 27

[0385] Sequence alignment of the mouse VH and VL sequences of v39093 (see Table 1.1) to respective human germline sequences identified IGHVl-69*01 and IGKVl-39*01 as the closest, as well as most frequent, human germline sequences. In addition, IGHJ4*01 and IGKJ2*01 joining region germline sequences, respectively, were selected. CDR sequences according to the AbM definition (see Table 3.1) were ported onto the framework of these selected human germline sequences as shown in Fig. 1 to generate the starting humanized VH and VL sequences Hl and LI, respectively. Back mutations to mouse residues in the resultant sequences at positions judged likely to be important for the retention of binding affinity to antigen, PTK7, were included creating several humanized sequences in which generated sequences were built on the previous sequence, and where the first humanized sequence (i.e. Hl or LI) contained no back mutations. CDR sequences of the parent antibody as defined by the AbM numbering system were not altered in any of the humanized sequences.

[0386] This process resulted in four variable heavy chain humanized sequences and three variable light chain humanized sequences. Full heavy chain sequences containing humanized heavy chain variable domain (VH) and hlgGl heavy chain constant domains (CHI, hinge, CH2, CH3), and full light chain sequence containing humanized light chain variable domain (VL) and human kappa light chain constant domain (kappa CL) were assembled. Monoclonal antibody (mAh) variants were then assembled such that each of the humanized heavy chains was paired with each of the humanized light chains to provide 12 humanized variants to be evaluated experimentally.3.1 Production of Humanized Antibody Constructs

[0387] Each of the 12 humanized antibody constructs was produced in full-size antibody (FSA) format containing two identical full-length heavy chains and two identical kappa light chains.

[0388] The full-length heavy chain contained the human CHl-hinge-CH2-CH3 domain sequences of IGHGl*01 (SEQ ID NO: 28; see Table 3.2). The light chain contained the human kappa CL sequence of IGKC*01 (SEQ ID NO: 29; see Table 3.2).Table 3.2: Human Constant Heavy and Light Chain SequencesSEQ Region Sequence ID NO CHl-hinge- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG 28 CH2-CH3 ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP (IGHGl*01) SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPG CHI ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG 107 (IGHGl*01) ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKV CL (kappa) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD 29 (IGKC*01) NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0389] Each of the humanized VH domain sequences was appended to the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 to provide four humanized full heavy chain sequences. Each of the humanized VL domain sequences was appended to the human kappa CL sequence of IGKC*01 to provide three humanized light chain sequences. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0390] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 6) (Barash et al., ibid.) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA. Sequences of the humanized VH and VL are provided in Table 3.3 below. CDR sequences of the humanized VH and VL are provided in Fig. 24.Table 3.3: Amino Acid Sequences of Humanized VH and VL SequencesSEQ Name Amino Acid Sequence ID NO QVQLVQSGAEVKKPGSSVKVSCKASGYALSRSWMNWVRQAPGQGLHl EWMGRIYSGDGDIHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAV 30 YYCARGSSPFDIWGQGTLVTVSS QVQLVQSGAEVKKPGSSVKVSCKASGYALSRSWMNWVRQAPGQGLH2 EWMGRIYSGDGDIHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAV 31 YYCAWGSSPFDIWGQGTLVTVSS QVQLVQSGAEVKKPGSSVKISCKASGYALSRSWMNWVRQAPGQGLEH3 WIGRIYSGDGDIHYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVY 32 YCAWGSSPFDIWGQGTLVTVSS EVQLVQSGAEVKKPGSSVKISCKASGYALSRSWMNWVRQAPGQGLEH4 WIGRIYSGDGDIHYAQKFQGRATLTADKSTNTAYMELSSLRSEDTAV 33 YYCAWGSSPFDIWGQGTLVTVSS DIQMTQSPSSLSASVGDRVTITCRASQSISDYLHWYQQKPGKAPKLLIY LI YASQSIPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNGHSFPWTFG 34 QGTKLEIK DIQLTQSPSSLSASVGDRVTITCRASQSISDYLHWYQQKPGKAPKLLIKL2 YASQSIPGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNGHSFPWTFG 35 QGTKLEIK DIQLTQSPSSLSASVGDRVTLTCRASQSISDYLHWYQQKPGKAPKLLIKL3 YASQSIPGIPSRFSGSGSGTDFTLTISSVQPEDFATYYCQNGHSFPWTFG 36QGTKLEIK

[0391] Transfection Conditions: The 12 humanized variants were transiently expressed in CHO 3E7 cells following the protocol described in Example 1, with the modification that the transfections were performed in 200 mL culture volumes.

[0392] Protein A Affinity and Preparative SEC: Protein A affinity chromatography and preparative SEC were conducted following the protocol described in Example 1, with the modification that samples either underwent preparative SEC or were buffer exchanged into DPBS using Zeba™ spin desalting columns (Thermo Scientific).

[0393] Final yields for the 12 humanized antibody variants were similar, ranging from approximately 15-18 mg (from 200 ml culture).

[0394] Caliper: Purity of samples was assessed by electrophoresis under non-reducing (NR) and reducing (R) conditions following the protocols as described in Example 1.

[0395] NR Caliper results reflected predominantly single species corresponding to full-size antibody and R Caliper reflected intact heavy and light chains.

[0396] UPLC-SEC: Species homogeneity of the final humanized antibody variant samples was assessed by UPLC-SEC following the protocol described in Example 1 with the modification that the Waters Acquity BEH200 SEC column was set to 25 °C or 30°C.

[0397] UPLC-SEC profiles were reflective of high species homogeneity. For all variants, ~98-99% monomeric species purity was determined.

[0398] Activity: The humanized antibodies were submitted to a preliminary screen to assess cytotoxicity and all variants except those comprising the humanized heavy chain Hl showed comparable or better activity to that of the chimeric parental antibody, v39093.3.2 Binding of Humanized Antibody Constructs to Human and Cynomolgus PTK7

[0399] Three of the humanized antibodies (H2_L1, H2_L2 and H3_L1) were selected for further evaluation. The ability of the selected humanized antibodies to bind to human and cynomolgus monkey PTK7 was assessed by flow cytometry using CHO-S cells as described below. Cofetuzumab (vl9287) was used as a positive control, and palivizumab (anti-RSV) (v22277) was used as a negative control.

[0400] Briefly, for expression of human PTK7, CHO-S cells were stably transfected with a linearized pTT5 -based human PTK7 plasmid, 0.5 pg DNA per 1 million cells, and FACS sorted to isolate human PTK7 expressors. For expression of cynomolgus PTK7, CHO-S cells were transiently transfected for ~24 hours with a pTT5 -based expression plasmid encoding cynomolgus monkey PTK7, 0.5 pg DNA per 1 million cells, using the Neon™ Transfection System (Thermo Fisher Scientific Inc., Waltham, MA), to transiently express cynomolgus monkey PTK7.

[0401] Cells were seeded at 50,000 cells / well in V-bottom 96-well plates and treated with antibody overnight at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Eabs, West Grove, PA; Cat. No. 109-605-098) at 4°C for 30 min. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, FranklinLake, NJ) with 1,000 minimum events collected per well. The AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live singlet cell population for each primary antibody using FlowJo™ v8 software (BD Biosciences, Franklin Lake, NJ). The Bmax and apparent Kd of each antibody was calculated using GraphPad Prism Version 9 (GraphPad Software, San Diego, CA).

[0402] The results of the dose-response curve for the humanized 13G11 anli-PJ’K7 antibodies are shown in Table 3.4 and Fig. 2. The positive control vl9287 showed binding to human PTK7 and cynomolgus monkey PTK7 on transfected CHO-S cells, as expected. No binding by negative control v22277 was observed, as expected. Humanized variants v39112, v39113 and v39115 bound similarly to both human and cynomolgus monkey PTK7, yielding apparent Kd values and Bmax values within 2-fold of each other. In comparison to parental chimera, v39093, humanized variants showed lower Bmax values for both human and cynomolgus monkey PTK7, but comparable apparent Kd to human PTK7 and superior apparent Kd to cynomolgus monkey PTK7.Table 3.4: Dose-Response Binding of Chimeric and Humanized 13G11 Antibodies to Human and Cynomolgus Monkey PTK7Bmax Apparent Kd (nM) VariantCommon NameNo. CHO-S CHO-S CHO-S CHO-S Hu PTK7 Cyno PTK7 Hu PTK7 Cyno PTK7 v39093 13G11 chimera 42793 1181 0.97 56.36 v39112 13G11 H2_L1 18958 696.3 0.83 8.90 v39113 13G11 H2_L2 23205 783.0 1.07 14.27 v39115 13G11 H3_L1 23064 782.5 0.83 8.52 V19287 Cofetuzumab 18092 945.0 1.18 21.93 V22277 Palivizumab IC IC IC IC*IC=Incomplete curve

[0403] The humanized antibody v39115 was selected for further evaluation. The CDR, VH, VL and full-length heavy and light chain sequences for this antibody are summarized in Table 3.5.Table 3.5: CDR, VH, VL and Full-length Heavy and Light Chain Sequences for v39115Region Sequence SEQ ID NOHeavy chain (full) QVQLVQSGAEVKKPGSSVKISCKASGYALSRSWMNWVRQAPGQ 96 GEEWIGRIYSGDGDIHYAQKFQGRATETADESTSTAYMEESSERS EDTAVYYCAWGSSPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DEETKNQVSETCEVKGFYPSDIAVEWESNGQPENNYKTTPPVED SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG VH QVQLVQSGAEVKKPGSSVKISCKASGYALSRSWMNWVRQAPGQ 32 GLEWIGRIYSGDGDIHYAQKFQGRATLTADESTSTAYMELSSLRS EDTAVYYCAWGSSPFDIWGQGTLVTVSS HCDR1 (IMGT) GYALSRSW 7 HCDR2 (IMGT) IYSGDGDI 8 HCDR3 (IMGT) AWGSSPFDI 9 HCDR1 (Kabat) RSWMN 10 HCDR2 (Kabat) RIYSGDGDIHYAQKFQG 11 HCDR3 (Kabat) GSSPFDI 12 HCDR1 (Chothia) GYALSRS 13 HCDR2 (Chothia) YSGDGD 14 HCDR3 (Chothia) GSSPFDI 12 HCDR1 (AbM) GYALS RSWMN 15 HCDR2 (AbM) RIYSGDGDIH 16 HCDR3 (AbM) GSSPFDI 12 HCDR1 (Contact) SRSWMN 17 HCDR2 (Contact) WIGRIYSGDGDIH 18 HCDR3 (Contact) AWGSSPFD 19 Light chain (full) DIQMTQSPSSESASVGDRVTITCRASQSISDYEHWYQQKPGKAPK 97 EEIYYASQSIPGVPSRFSGSGSGTDFTETISSEQPEDFATYYCQNG HSFPWTFGQGTKEEIKRTVAAPSVFIFPPSDEQEKSGTASVVCEEN NFYPREAKVQWKVDNAEQSGNSQESVTEQDSKDSTYSESSTETE SKADYEKHKVYACEVTHQGESSPVTKSFNRGEC VL DIQMTQSPSSESASVGDRVTITCRASQSISDYEHWYQQKPGKAPK 34 EEIYYASQSIPGVPSRFSGSGSGTDFTETISSEQPEDFATYYCQNG HSFPWTFGQGTKEEIK LCDR1 (IMGT) QSISDY 20Region Sequence SEQ ID NO LCDR2 (IMGT) YAS 21 LCDR3 (IMGT) QNGHSFPWT 22 LCDR1 (Kabat) RASQSISDYLH 23 LCDR2 (Kabat) YASQSIP 24 LCDR3 (Kabat) QNGHSFPWT 22 LCDR1 (Chothia) RASQSISDYLH 23 LCDR2 (Chothia) YASQSIP 24 LCDR3 (Chothia) QNGHSFPWT 22 LCDR1 (AbM) RASQSISDYLH 23 LCDR2 (AbM) YASQSIP 24 LCDR3 (AbM) QNGHSFPWT 22 LCDR1 (Contact) SDYLHWY 25 LCDR2 (Contact) LLIYYASQSI 26 LCDR3 (Contact) QNGHSFPW 27EXAMPLE 4: HUMANIZATION OF ANTI-PTK7 ANTIBODY 14D08 AND ASSESSMENT OF BINDING TO HUMAN AND CYNOMOLGUS PTK7

[0404] The chimeric anti-human PTK7 antibody construct, variant v39090, generated as described in Example 1, was humanized as described below. The CDR sequences of the chimeric antibody v39090, which are the same as those of the parental anti-PTK7 antibody 14D08, are provided in Table 4.1.Table 4.1: CDR Sequences of the Chimeric Antibody v39090 (Anti-PTK7 Antibody 14D08)SequenceNumbering SEQ SEQ SEQ System Heavy Chain ID Heavy Chain ID Heavy Chain ID CDR1 CDR2 CDR3NO NO NO IMGT GFSLSSYG 37 IWSDGST 38 ARHNGYYAMDY 39 Kabat SYGVH 40 LIWSDGSTTYNS 41 HNGYYAMDY 42 ALKSChothia GFSLSSY 43 WSDGS 44 HNGYYAMDY 42 AbM GFSLSSYGVH 45 LIWSDGSTT 46 HNGYYAMDY 42Contact SSYGVH 47 WLVLIWSDGSTT 48 ARHNGYYAMD 49SEQ SEQ SEQLight Chain Light Chain Light ChainID ID ID CDR1 CDR2 CDR3NO NO NO IMGT KSISKY 50 SGS 51 QQHNEYPLT 52 Kabat RASKSISKYLA 53 SGSTLQF 54 QQHNEYPLT 52 Chothia RASKSISKYLA 53 SGSTLQF 54 QQHNEYPLT 52 AbM RASKSISKYLA 53 SGSTLQF 54 QQHNEYPLT 52 Contact SKYLAWY 55 LLIYSGSTLQ 56 QQHNEYPL 57

[0405] Sequence alignment of the mouse VH and VL sequences of v39090 (see Table 1.1) to respective human germline sequences identified IGHV3-23*01 and IGKVl-39*01 as the closest, as well as most frequent, human germline sequences. In addition, IGHJ4*01 and IGKJ2*01 joining region germline sequences, respectively, were selected. CDR sequences according to the AbM definition (see Table 4.1) were ported onto the framework of these selected human germline sequences as shown in Fig. 3 to generate the starting humanized VH and VL sequences Hl and LI, respectively. Back mutations to mouse residues in the resultant sequences at positions judged likely to be important for the retention of binding affinity to antigen, PTK7, were included creating several humanized sequences in which generated sequences were built on the previous sequence, and where the first humanized sequence (i.e. Hl or LI) contained no back mutations. CDR sequences of the parent antibody as defined by the AbM numbering system were not altered in any of the humanized sequences.

[0406] This process resulted in four variable heavy chain humanized sequences and three variable light chain humanized sequences. Full heavy chain sequences containing humanized heavy chain variable domain (VH) and hlgGl heavy chain constant domains (CHI, hinge, CH2, CH3), and full light chain sequence containing humanized light chain variable domain (VL) and human kappa light chain constant domain (kappa CL) were assembled. Monoclonal antibody (mAh) variants were then assembled such that each of the humanized heavy chains was paired with each of the humanized light chains to provide 12 humanized variants to be evaluated experimentally.4.1 Production of Humanized Antibody Constructs

[0407] Each of the 12 humanized antibody constructs was produced in full-size antibody (FSA) format containing two identical full-length heavy chains and two identical kappa light chains.

[0408] The full-length heavy chain contained the human CHl-hinge-CH2-CH3 domain sequences of IGHGl*01 (SEQ ID NO: 28; see Table 3.2 in Example 3). The light chain contained the human kappa CL sequence of IGKC*01 (SEQ ID NO: 29; see Table 3.2 in Example 3).

[0409] Each of the humanized VH domain sequences was appended to the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 to provide four humanized full heavy chain sequences. Each of the humanized VL domain sequences was appended to the human kappa CL sequence of IGKC*01 to provide three humanized light chain sequences. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0410] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 6) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842) and the heavy chain clone terminating at residue G446 (EU numbering) of the CH3 domain were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA. Sequences of the humanized VH and VL are provided in Table 4.3 below. CDR sequences of the humanized VH and VL are provided in Fig. 24.Table 4.3: Amino Acid Sequences of Humanized VH and VL SequencesName Amino Acid Sequence SEQ ID NOHl EVQLLESGGGLVQPGGSLRLSCAASGFSLSSYGVHWVRQAPGKGLE 58 WVSLIWSDGSTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCAKHNGYYAMDYWGQGTLVTVSSH2 EVQLLESGGGLVQPGGSLRLSCAVSGFSLSSYGVHWVRQAPGKGLE 59 WVSLIWSDGSTTYADSVKGRFTISKDNSKNTVYLQMNSLRAEDTAV YYCARHNGYYAMDYWGQGTLVTVSSH3 EVQLLESGGGLVQPGGSLRISCAVSGFSLSSYGVHWVRQAPGKGLE 60 WLVLIWSDGSTTYADSLKGRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARHNGYYAMDYWGQGTLVTVSSName Amino Acid Sequence SEQ ID NOH4 QVQLLESGGGLVQPGGSLRISCAVSGFSLSSYGVHWVRQAPGKGLE 61 WLVLIWSDGSTTYADSLKGRLTISKDNSKNTVYLQMNSLRAEDTAV YYCARHNGYYAMDYWGQGTLVTVSS LI DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLI 62 YSGSTLQFGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPL TFGQGTKLEIKL2 DIQLTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLI 63 YSGSTLQFGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLT FGQGTKLEIKL3 DIVLTQSPSSLSASVGDRITITCRASKSISKYLAWYQEKPGKTNKLLIY 64 SGSTLQFGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQGTKLELK

[0411] The same protocols as described in Example 3 were used for transfection and purification of the 12 humanized antibodies and their characterization by Caliper and UPLC-SEC.

[0412] Final yields for the 12 humanized antibody variants were similar, ranging from approximately -9-15 mg (from 200 ml culture). Non-reducing (NR) and reducing (R) Caliper reflected single species corresponding to full-size antibody and intact heavy and light chains.

[0413] UPLC-SEC profiles were reflective of high species homogeneity, for all variants -95-100% monomeric species purity was determined.

[0414] Activity: The humanized antibodies were submitted to a preliminary screen to assess cytotoxicity and all variants showed comparable or better activity to that of the chimeric parental antibody, v39090.4.2 Binding of Humanized Antibody Constructs to Human and Cynomolgus PTK7

[0415] The ability of the selected humanized anti-PTK7 antibodies (H1_L1, H1_L3 and H2_L1) to human and cynomolgus monkey PTK7 was assessed by flow cytometry using CHO-S cells following the protocol described in Example 3. Cofetuzumab (vl9287) was used as a positive control, and palivizumab (anti-RSV) (v22277) was used as a negative control.

[0416] The results of the dose-response curve for the selected humanized 14D08 anti-PTK7 antibodies are shown in Table 4.4 and Figure 4.

[0417] The positive control vl9287 showed binding to human PTK7 and cynomolgus monkey PTK7 on transfected CHO-S cells, as expected. No binding by negative control v22277 was observed, as expected. Humanized variants v39133, v39135 and v39136 bound to human and cynomolgus monkey PTK7 similarly to parental chimera v39090, yielding apparent Kd values and Bmax values within 2-fold.Table 4.4: Dose-response Binding of Chimeric and Humanized 14D08 Antibodies to Human and Cynomolgus Monkey PTK7Bmax Apparent Kd (nM) VariantCommon NameNo. CHO-S CHO-S CHO-S CHO-S Hu PTK7 Cyno PTK7 Hu PTK7 Cyno PTK7 v39090 14D08 chimera 62477 895.1 1.23 0.68 v39133 14D08 H1_L1 57317 937.1 0.64 0.46 v39135 14D08 H1_L3 48261 965.2 0.74 0.51 v39136 14D08 H2_L1 43588 1008 1.06 0.65 V19287 Cofetuzumab 18092 945.0 1.18 0.43 V22277 Palivizumab IC IC IC IC*IC=Incomplete curve

[0418] The humanized antibody v39133 was selected for further evaluation. The CDR, VH, VL and full-length heavy and light chain sequences for this antibody are summarized in Table 4.5.Table 4.5: CDR, VH, VL and Full-length Heavy and Light Chain Sequences for v39133 Region Sequence SEQ ID NOHeavy chain (full) EVQEEESGGGEVQPGGSERESCAASGFSESSYGVHWVRQAPGKG 98 LEWVSLIWSDGSTTYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKHNGYYAMDYWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGEYSESSVVTVPSSSEGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEEEGGPSVFEFPPKPKDTEMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVETV EHQDWENGKEYKCKVSNKAEPAPIEKTISKAKGQPREPQVYTEP PSRDEETKNQVSETCEVKGFYPSDIAVEWESNGQPENNYKTTPP VEDSDGSFFEYSKETVDKSRWQQGNVFSCSVMHEAEHNHYTQK SESESPGRegion Sequence SEQ ID NO VH EVQLLESGGGLVQPGGSLRLSCAASGFSLSSYGVHWVRQAPGKG 58 LEWVSLIWSDGSTTYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKHNGYYAMDYWGQGTLVTVSS HCDR1 (IMGT) GFSLSSYG 37 HCDR2 (IMGT) IWSDGST 38 HCDR3 (IMGT) AKHNGYYAMDY 90 HCDR1 (Kabat) SYGVH 40 HCDR2 (Kabat) LIWSDGSTTYADSVKG 92 HCDR3 (Kabat) HNGYYAMDY 42 HCDR1 (Chothia) GFSLSSY 43 HCDR2 (Chothia) WSDGS 44 HCDR3 (Chothia) HNGYYAMDY 42 HCDR1 (AbM) GFSLSSYGVH 45 HCDR2 (AbM) LIWSDGSTT 46 HCDR3 (AbM) HNGYYAMDY 42 HCDR1 (Contact) SSYGVH 47 HCDR2 (Contact) WVSLIWSDGSTT 93 HCDR3 (Contact) AKHNGYYAMD 91 Light chain (full) DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPK 99 LLIYSGSTLQFGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQH NEYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VL DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPK 62 LLIYSGSTLQFGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQH NEYPLTFGQGTKLEIK LCDR1 (IMGT) KSISKY 50 LCDR2 (IMGT) SGS 51 LCDR3 (IMGT) QQHNEYPLT 52 LCDR1 (Kabat) RASKSISKYLA 53 LCDR2 (Kabat) SGSTLQF 54 LCDR3 (Kabat) QQHNEYPLT 52 LCDR1 (Chothia) RASKSISKYLA 53 LCDR2 (Chothia) SGSTLQF 54Region Sequence SEQ ID NO LCDR3 (Chothia) QQHNEYPLT 52 LCDR1 (AbM) RASKSISKYLA 53 LCDR2 (AbM) SGSTLQF 54 LCDR3 (AbM) QQHNEYPLT 52 LCDR1 (Contact) SKYLAWY 55 LCDR2 (Contact) LLIYSGSTLQ 56 LCDR3 (Contact) QQHNEYPL 57EXAMPLE 5: FUNCTIONAL CHARACTERIZATION OF HUMANIZED ANTI-PTK7 ANTIBODIES – BINDING TO HUMAN AND CYNOMOLGUS MONKEY PTK7 ON WHOLE CELLS

[0419] The ability of the humanized anti-PTK7 monospecific antibodies, v39115 (13G11) and v39133 (14D08) to bind to human and cynomolgus monkey PTK7 was assessed by flow cytometry using human and cynomolgus monkey PTK7 -transfected CHO-S cells, and human cancer cell lines HCC1569 (breast carcinoma) and H446 (lung carcinoma) expressing endogenous PTK7, as described below. Anti-PTK7 antibody cofetuzumab (vl9287) and a high-affinity monospecific anti-PTK7 antibody, v38332, were included as positive controls. Palivizumab (anti-RSV) (v21995) was included as a negative control.

[0420] HCC1569 cells express endogenous PTK7 at high level and H446 cells express PTK7 at moderate level, as determined by surface protein quantification as described in Example 16.

[0421] Briefly, CHO-S cells were transiently transfected with a pTT5-based expression plasmid encoding human or cynomolgus monkey PTK7, 0.5 pg DNA per 1 million cells, using the Neon™ Transfection System (Thermo Fisher Scientific Corp., Waltham, MA), and incubated at 37°C / 5% CO2 / 110 rpm for 24 hours to transiently express human or cynomolgus monkey PTK7. HCC1569 and H446 cells were cultured in complete growth media (RPMI 1640 ATCC modification, Thermo Fisher Scientific, Waltham, MA, Cat. No. A1049101) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, Cat. No. 12483-020) and incubated overnight under standard culturing conditions (37°C / 5% CO2) to allow attachment. Cells were seeded at 50,000cells / well in a 96-well V-bottom plate (Sarstedt, Numbrecht, Germany, Cat. No. 82.1583.001) and treated with test articles for 24 hours at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA, Cat. No. 109-605-098) at 4°C for 1 hour. Following staining and washing, fluorescence was detected by flow cytometry on BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647 / APC- A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live singlet cell population for each primary antibody using FlowJo™ v8 software (BD Biosciences, Franklin Lake, NJ). The Bmax and Kd of each primary antibody were calculated using GraphPad Prism Version 9 (GraphPad Software, La Jolla, CA).

[0422] The binding results for v39115, v39133, v38332 and cofetuzumab (vl9287) are shown in Table 5.1 and Figure 5. On human PTK7 transfected CHO-S cells, variants v39115, v39133, v38332 and cofetuzumab showed comparable binding Kd. Variant v39115 and cofetuzumab showed lower binding Bmax than variants v39133 and v38332 against human PTK7 on transfected CHO-S cells. On cynomolgus monkey PTK7 transfected CHO-S cells, and HCC1569 and H446 cell lines, variants v39115, v39133, v38332 and cofetuzumab demonstrated comparable binding Kd and Bmax. Negative control palivizumab (v21995) did not bind to any cell line, as expected.Table 5.1: Binding of Anti-PTK7 Monospecific Antibodies to Human and Cynomolgus Monkey PTK7CHO-S Hu PTK7 CHO-S Cyno HCC1569 H446PTK7SampleApp Kd1App Kd App Kd App Kd Bmax Bmax Bmax Bmax(nM) (nM) (nM) (nM) v39115 13,733 0.23 1,050 0.01 9,028 0.16 6,205 0.07 v39133 28,825 0.57 1,423 0.04 8,189 0.13 5,699 0.08 v38332 32,066 0.77 1,566 0.07 9,631 0.17 6,882 0.09 cofetuzumab 8,024 0.16 1,249 0.04 7,682 0.13 6,417 0.09 palivizumabNB2NB NB NB NB NB NB NB1App Kd= apparent Kd2NB = no binding (apparent Kdvalue greater than the highest antibody testing concentration (>200 nM)) EXAMPLE 6: FUNCTIONAL CHARACTERIZATION OF ANTI-PTK7 ANTIBODIES - INTERNALIZATION

[0423] The PTK7-mediated internalization capability of the monospecific antibodies v39115 (13G11) and v39133 (14D08) in two PTK7-expressing cell lines was determined by flow cytometry as described below. The anti-PTK7 antibody cofetuzumab (vl9287) and a high-affinity monospecific anti-PTK7 antibody, v38332, were included as positive controls. The anti-RSV antibody palivizumab was used as a negative control.

[0424] Cell lines used were HCC1569 (breast carcinoma) and H446 (lung carcinoma). HCC1569 cells express endogenous PTK7 at high level and H446 cells express PTK7 at moderate level, as determined by surface protein quantification as described in Example 16.

[0425] Briefly, antibodies were fluorescently labeled by coupling to a Fab fragment AF488 conjugate targeting human IgG Fc (Jackson ImmunoResearch Labs, West Grove, PA; Cat. No.109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No. 10010-023), for 24 hours at 4°C. Cells were seeded at 50,000 cells / well in their respective media in 48-well plates (HCC1569 and H446 cells in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. A1049101) supplemented with 10% fetal bovine serum) and incubated overnight under standard culturing conditions (37°C / 5% CO2) to allow attachment. Coupled antibodies were added to cells the following day at 10 nM and incubated under standard culturing conditions for 4 hours to allow for internalization. An untreated control (cells incubated with complete growth medium only, no test article) was included for each cell line at each treatment time point. Following incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A- 11094) at 100 nM for 30 minutes at 4°C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF488 / FITC-A GeoMean (fluorescence signal area geometric mean, proportional to anti-Human Fab AF488 labelling) was calculated for the live single cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ), normalized to the fluorescence signal of the untreated control, and plotted using GraphPad Prism Version 9 (GraphPad Software, Fa Jolla, CA).

[0426] The results for the 10 nM test concentration are shown in Table 6.1 and Fig. 6A-B. Positive control antibody cofetuzumab (vl9287) showed internalization in both HCC1569 andH446 cell lines, as expected, and antibody v38332 showed comparable internalization to cofetuzumab in both HCC1569 and H446 cell lines. Both anti-PTK7 antibodies v39115 and v39133 showed increased internalization compared to palivizumab negative control in both cell lines evaluated. Antibody v39133 showed lower internalization than cofetuzumab in both HCC1569 and H446 cell lines. Antibody v39115 showed slightly lower internalization than cofetuzumab in the HCC1569 cell line and slightly higher internalization than cofetuzumab in the H446 cell line.Table 6.1: Internalization of Anti-PTK7 AntibodiesInternalized Fluorescence(Fold-Over Palivizumab)Antibody (10 nM)HCC1569 H446v39115 12.2 12.1v39133 7.2 7.5v38332 17.2 10.8Cofetuzumab 18.6 10.8Palivizumab 1.0 1.0EXAMPLE 7: DEVELOPABILITY ASSESSMENT OF ANTI-PTK7 ANTIBODIES

[0427] The isoelectric point, propensity for self-aggregation, and non-specific binding of the anti-PTK7 antibodies v39115 and v39133 were determined. Assessment of these properties can provide a preliminary indication of the developability of antibodies. Cofetuzumab (vl9287) and trastuzumab (vl7079) were used as benchmark comparisons. The isoelectric point was measured by capillary isoelectric focusing (cIEF), the propensity for self-aggregation was measured by affinity-capture self-interaction nanoparticle spectroscopy (AC-SINS) and non-specific (NS) binding was measured by NS-ELISA, as described below.Capillary isoelectric focusing ( cIEF)

[0428] cIEF was conducted using a Maurice™ C system, System Suitability Kit and Method Development Kit (ProteinSimple Inc., San Jose, CA). System suitability standard, fluorescence calibration standard, cartridge and samples were prepared according to manufacturer’srecommendations. The capillary was automatically calibrated with a fluorescence standard preconditioned with Maurice™ cIEF System Suitability Kit to ensure the capillary was functioning correctly. The antibody samples were diluted to a concentration of 0.5 mg / mL in a final volume of 40 pL in Gibco™ Distilled Water, and mixed Maurice™ cIEF Method Development Kit Samples. The samples were then vortexed, centrifuged and the supernatant pipetted into individual wells of a 96-well plate. All electropherograms were detected with UV absorbance at 280 nm and protein native fluorescence. All data analyses were performed using manufacturer’s software Compass for iCE (ProteinSimple Inc.). The Compass software aligns each electropherogram using the pl markers so that the x-axis is displayed as a normalized pl for each injection. The pl values were determined for the main isoform for each of the variants and benchmark controls.Affinity-capture self -inter action nanoparticle spectroscopy (AC-SINS) assay

[0429] The AC-SINS assay was carried out in a 384-well plate format. Initial ly, 20 nm gold nanoparticles (Ted Pella Inc., Redding, CA; Cat. No. 15705) washed with 0.22 pm filtered Gibco™ Distilled Water were coated with a mixture of capture antibody, 80% AffiniPure™ Goat Anti-Human IgG (H+L) (Jackson ImmunoResearch Laboratories; Cat. No. 109-005-088), and the non-capture antibody, 20% ChromPure™ Goat IgG, whole molecule (Jackson ImmunoResearch Laboratories; Cat. No. 005-000-003), that were initially buffer exchanged into 20 mM sodium acetate pH 4.3 and diluted to 0.4 mg / mL. The mixture of gold nanoparticles, capture antibody and non-capture antibody was incubated in the dark for 18h at room temperature. Sites unoccupied on the gold nanoparticles were blocked with 1 pM thiolated polyethylene glycol (2 kD) in 20 mM sodium acetate, pH 4.3, to a final concentration of 0.1 pM, followed by Ih incubation at room temperature. The coated nanoparticles were then concentrated by centrifugation at 21,000 g for 7 min, at 8°C. 95% of the supernatant was removed and the gold pellet was resuspended in the remaining buffer. 5 pL of concentrated nanoparticles were added to 45 pL of antibody at 0.05 mg / mL in Gibco™ PBS pH 7.4 in a 384-well plate. The coated nanoparticles were incubated with the test antibody for 4h at room temperature in the dark. The absorbance was scanned from 450-700nm at 1 nm increments, and a Microsoft Excel macro was used to identify the max absorbance, smooth the data, and fit the data using a second-order polynomial.

[0430] The Alambda (nm) was calculated based on the smoothed max absorbance of the average blank (PBS alone) subtracted from the smoothed max absorbance of the antibody sample todetermine the antibody AC-SINS score. Antibody-antibody interactions directly correlate with the shift in maximum absorbance wavelength of gold nanoparticles coated with the test antibody. A cut-off of Alambda lOnm was set as high self-aggregation propensity of the antibody, based on the literature (Bailly, M., et al., 2020, mAbs, 12(1); Jain, T., et al., 2017, PNAS, 1114(5): 944-949). Non-Specific (NS)-ELISA

[0431] NS-ELISA was used to measure the propensity of the antibodies to bind to a range of biomolecules to emulate the undesirable non-specific interactions to biological matrices in vivo as described below.

[0432] NS-ELISA was carried out in a Corning® 96-well EIA / RIA Easy Wash™ Clear Flat Bottom Polystyrene High Bind Microplate coated overnight at 4°C with 50 mL of heparin (Sigma-Aldrich, St. Louis, MI; Cat. No. H3149) diluted with 50 mM sodium carbonate pH 9.6 to a final concentration of 250 pg / mL. The plate was incubated for 2 days at room temperature, wells that were coated with heparin were not covered to allow for air drying. Insulin (Sigma-Aldrich, St. Louis, MI; Cat. No. 19278) and KLH (Sigma-Aldrich, Cat. No. H8283) were each diluted with 50 mM sodium carbonate pH 9.6 to a final concentration of 5 pg / mL. ssDNA (Sigma- Aldrich, Cat. No. D8899) and dsDNA (Sigma-Aldrich, Cat. No. D4553) were diluted with PBS pH7.4 to a final concentration of 10 pg / mL. 50 pL each of insulin, KLH, dsDNA and ssDNA were added to a 96-well plate, followed by the incubation at 37°C for 2h. The coating materials were removed, and the plate was blocked with 200 pL of PBS pH7.4, 0.1 % Tween® 20, and incubated for Ih at room temperature with shaking at 200rpm. The plate was washed 3 times with PBS pH7.4, 0.1% Tween® 20. 50 pL of each antibody at 100 nM (15 mg / mL) in PBS pH 7.4, 0.1% Tween® 20 was added in duplicate to the wells and incubated for Ih at room temperature with shaking at 200 rpm. Plates were washed three times with PBS pH7.4, 0.1% Tween® 20, and 50 pL of 50 ng / mL antihuman IgG horseradish peroxidase (HRP) (Thermo Fisher Scientific Inc., Waltham, MA; Cat. No. H10307) was added to each well. Plates were incubated for Ih at room temperature, with shaking at 200 rpm. The plate was washed three times with PBS pH7.4, 0.1% Tween® 20, and 100 pL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Cell Signaling Technology, Danvers, MA; Cat. No. 7004P6) added to each well. Reactions were stopped after approximately 10 minutes by adding 100 pL of 1 M HC1 to each well, and absorbance was read at 450 nm. Binding scores werecalculated as the ratio of the ELISA signal of the antibody to the signal of a well containing buffer instead of the primary antibody.

[0433] The results of all three assays are shown in Table 7.1. No flags were identified for the anti-PTK7 antibodies v39115 and v39133. Alambda (AC-SINS) and binding scores (NS-ELISA) for both v39115 and v39133 were similar to those of cofetuzumab (vl9287) and trastuzumab (vl7079). No extreme pl was detected for v39115 or v39133 by cIEF with values being similar to those for cofetuzumab (vl9287) and trastuzumab (vl7079).Table 7.1: Developability Assessment ResultscIEF AC-SINS NS-ELISAVariant CommonNo Name Alambdapl ssDNA KLH Insulin dsDNA Heparin (nm)13G11v39115 8.85 4.5 0.84 1.52 0.77 1.03 0.71 H3_L114D08v39133 9.05 5.0 0.95 2.49 1.39 0.91 0.94 H1_L1v 19287 Cofetuzumab 8.26 4.0 0.96 1.94 0.83 0.94 0.78 v 17079 Trastuzumab 9.00 4.5 1.03 1.84 1.11 0.84 0.69EXAMPLE 8: BINDING AFFINITY ASSESSMENT OF ANTI-PTK7 ANTIBODIES

[0434] SPR analysis was performed to characterize binding affinity of the anti-PTK7 antibodies v39115 and v39133 to human and cynomolgus monkey PTK7 antigens as described below. Cofetuzumab (vl9287) was used as a positive control.

[0435] Binding kinetics were carried out at 25 °C using a Cytiva Biacore™ T200 instrument (Cytiva, Marlborough, MA) with a Series S Sensor Chip CM5 (Cytiva; Cat. No. BR100399), which was amine-coupled with anti-human Fc antibody using the Cytiva anti-Fc immobilization kit (Cytiva; Cat. No. BR100839). The chip surface decorated with carboxyl groups was activated with 400 mM N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide (EDC) and 100 mM N-hydroxysuccinimide (NHS). The anti-Fc antibody was injected into flow cells 1 to 4 with a contact time of 420 secs at a flow rate of 10 µL / min for immobilization. The reaction was stopped with 1 M ethanolamine-HCl pH8.5, followed by a buffer wash step using the running buffer, lx HBSEP+ buffer (lOmM HEPES, 150mM NaCl, 3mM EDTA, 0.005% Tween- 20). For affinity analysis, each test article at a concentration of 4 pg / niL was injected into flow cells 2, 3 and 4 as binding ligand at a flow rate of 10 p L / inin for 20 sec. Test articles were captured by immobilized anti-Fc antibody. Flow cell 1 was used as a reference surface. Human PTK7 (ACROBiosystems, Newark, DE; Cat. No. PT7-H52H3) and cynomolgus PTK7 (ACROBiosystems; Cat. No. PT7-C52H3) were serially diluted 3-fold to 0.56-45 nM in lx HBS EP+ buffer. Each of the PTK7 antigens at different dilutions were flowed as analyte using single-cycle kinetics strategy. The flow rate of analyte was 50 uL / min with contact time of 120 sec. Surface regeneration was accomplished using lOmM glycine-HCl, pH 1.5 for 60 sec with a flow rate of 50 pL / min after each cycle. Binding constants were determined with the Biacore™ T200 Evaluation software (version 3.0; GE Healthcare Technologies Inc., Chicago, IL) and calculated using a 1:1 binding fit model.

[0436] The results of SPR binding analysis are shown in Table 8.1. The rate of complex formation is represented as the association constant kon, and the rate of complex dissociation is represented by koff. The affinity of the analyte for the ligand (the equilibrium dissociation constant, KD) is calculated from the kinetic association and dissociation rates (koff / kon). Variants v39115 and v39133 showed similar affinities (KD) for both cynomolgus and human PTK7 antigens, and exhibited comparable KD values for cynomolgus and human PTK7 to cofetuzumab (vl9287) at a nanomolar scale.Table 8.1: SPR Binding Analysis for Anti-PTK7 Antibodies for Human and Cynomolgus Monkey PTK7Common AntigenTest Article (1 / Ms) koff(1 / s) KD(nM) Name Species13G11 Human 1.28E+05 1.07E-03 8.42 v39115H3_L1 Cynomolgus 1.43E+05 1.16E-03 8.18 14D08 Human 9.40E+04 4.97E-04 5.32 v39133H1_L1 Cynomolgus 3.28E+05 1.76E-03 5.68Human 7.18E+04 4.67E-04 6.55 V19287 CofetuzumabCynomolgus 9.02E+04 3.93E-04 4.36EXAMPLE 9: DESIGN, PRODUCTION AND CHARACTERIZATION OF ANTI-PTK7 BIPARATOPIC ANTIBODY

[0437] A biparatopic antibody, v39794, comprising the anti-PTK7 paratopes from each of v39115 and v39133 was designed, produced and characterized as described below.9.1 Biparatopic Antibody Design

[0438] Identification of suitable anli-P'l’KV paratopes for inclusion in an anti-PTK7 biparatopic antibody was achieved through an extensive screening approach that involved first creating a library of 2304 biparatopic antibody candidates in chimeric Fab-scFv format using VH / VL pairs from 29 different mouse anti-PTK7 antibodies, including antibodies 13G11 and 14D08. The activity of the biparatopic antibody candidates in PTK7 binding assays and in vitro cytotoxicity assays was then assessed and six biparatopic antibodies that showed improved human PTK7 binding and improved potency in vitro compared to benchmark were selected and the VH / VL sequences were humanized (as described in Examples 3 and 4 for antibodies 13G11 and 14D08, respectively). The activity and biophysical characteristics of each of the humanized VH / VL pairs were assessed following the protocols described in Examples 5-8 for 13G11 and 14D08 leading to the selection of v39115 and v39133 as the lead anti-PTK7 paratopes for incorporation into the final biparatopic antibody v39794.

[0439] An IgG-like bispecific format was employed for the biparatopic antibody, v39794, containing two different full-size heavy chains (HC1 and HC2) and 2 different light chains (LC1 and LC2) (see Table 9.1). One heavy and light chain corresponded to the humanized v39115 (13G11 H3L1) paratope and the other heavy and light chain corresponded to the humanized v39133 (14D08 HILI) paratope. The variable domain sequences (VH and VL) for humanized 13G11 H3L1 and 14D08 HILI are provided in Examples 3 and 4 (Tables 3.3 and 4.3), respectively.

[0440] To ensure correct light and heavy chain pairing in the Fab portion of the biparatopic molecule, amino acid mutations were introduced into the CHI and CL domains of each paratope. These mutations are referred to as design 4478 and comprise (Kabat numbering):Q179R introduced into the first CHI (“CHI (C)”)Q124E_L135W_T178E_T180E introduced into the first CL (“CL (C)”), A139W_L143E_K145T_Q179E introduced into the second CHI (“CHI (D)”)Fl 16A_Q124R_L135V_T178R introduced into the second CE (“CL (D)”)

[0441] Where C and D denote the two different paratopes (13G11 and 14D08, respectively) present in biparatopic antibody. The sequences of CHI (C), CL (C), CHI (D) and CL (D) are provided in Table 9.2).

[0442] The CH3 domain of each heavy chain construct contained the mutations: T350V_L351Y_F405A_Y407V (chain A), or the mutations: T350V_T366L_K392L_T394W (chain B) (EU numbering) to ensure correct pairing of the heavy chains in the Fc region. The CH3 domain sequences are also provided in Table 9.2.

[0443] Full-length HC1, HC2, LC1 and LC2 amino acid sequences for v39794 are provided in Table 9.3.Table 9.1: Biparatopic Antibody CompositionVariant Format Description Domain CompositionNo.v39794 14D08 (HILI) x HC1: VH(13G11)-CH1(C)-HINGE- 13G11(H3L1) CH2-CH314D08%^ 13G11 HC2: VH(14DO8)-CH1(D)-HINGE- I CH2-CH3LC1: VL(13G11)-CL(C)LC2: VL(14D08)-CL(D)Table 9.2: CH and CL Domain Sequences used in Variant V397941Domain Sequence SEQ ID NO CHI (C) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL Q179R TSGVHTFPAVLRSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK 65VDKKV CHI (D) ASTKGPSVFPLAPSSKSTSGGTAWLGCEVTDYFPEPVTVSWNSGAL A139W_L143E_ TSGVHTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK 66 K145T_Q179E VDKKVHINGE EPKSCDKTHTCPPCP 67(IGHG1*O1)Domain Sequence SEQ ID NO CH2 (IGHG1*O1) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY 68 KCKVSNKALPAPIEKTISKAK CH3(IGHG1*O1)_ GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG T350V_L351Y_ QPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSL 69 F405A_Y407V SLSPG(chain A)CH3(IGHG1*O1)_ GQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNG T350V_T366L_ QPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSL 70 K392L_T394W SLSPG(chain B)CL (C) RTVAAPSVFIFPPSDEELKSGTASVVCWLNNFYPREIKVQWKVDN Q124E_L135W_ ALQSGNSQESVTEQDSKDSTYSLSSELELSKADYEKHKVYACEVT T178E_T180E (and HQGLSSPVTKSFNRGEC 71 A144I2)CL (D) RTVAAPSVAIFPPSDERLKSGTASVVCVLNNFYPREAKVQWKVDN F116A_Q124R ALQSGNSQESVTEQDSKDSTYSLSSRLTLSKADYEKHKVYACEVT 72 _L135V_T178R HQGLSSPVTKSFNRGEC1Mutations in CHI and CL domains use Kabat numbering and mutations in CH2 and CH3 domains use EU numbering2Modification included for mass separation purposesTable 9.3: Full-Length HC1, HC2, LC1 and LC2 Amino Acid Sequences for v39794 Name Amino Acid Sequence SEQ ID NO HC1 QVQLVQSGAEVKKPGSSVKISCKASGYALSRSWMNWVRQAPGQGLEW 73 IGRIYSGDGDIHYAQKFQGRATLTADESTSTAYMELSSLRSEDTAVYYC AWGSSPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLRSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG HC2 EVQLLESGGGLVQPGGSLRLSCAASGFSLSSYGVHWVRQAPGKGLEWV 74 SLIWSDGSTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KHNGYYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAWLG CEVTDYFPEPVTVSWNSGALTSGVHTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQP ENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG LC1 DIQMTQSPSSLSASVGDRVTITCRASQSISDYLHWYQQKPGKAPKLLIYY 75 ASQSIPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNGHSFPWTFGQ GTKLEIKRTVAAPSVFIFPPSDEELKSGTASVVCWLNNFYPREIKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSELELSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC LC2 DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYS 76 GSTLQFGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQ GTKLEIKRTVAAPSVAIFPPSDERLKSGTASVVCVLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSRLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

[0444] Protein sequences for heavy and light chains were assembled according to Tables 9.1 and 9.2 and Tables 3.3 and 4.3 from Examples 3 and 4. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0445] Heavy chain vector inserts comprising the signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 6) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842) were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA.9.2 Production and QC of Biparatopic Antibody

[0446] The biparatopic antibody v39794 was produced in CHO cells, then purified by protein A chromatography and preparative SEC. The resultant sample was characterized for homogeneity (as determined by UPLC-SEC), chain intactness (as determined by Caliper and LC-MS) and degree of heterodimer pairing (i.e. bispecific purity) as determined by LC-MS.

[0447] Cloning and Transfection Conditions: The biparatopic antibody was transiently expressed in CHO 3E7 cells.

[0448] Briefly, CHO-3E7 cells were cultured in FreeStyle™ F17 expression medium (Gibco; Cat No. A- 1383501) supplemented with 4 mM L-glutamine (HyClone / Cytivia; Cat. No. SH30034.01) and 0.1% Pluronic F-68™ (Gibco; Cat. No. 24040-032) at 37°C. A transfection mixture of 3.5mg DNA (1.75mg DNA coding for the biparatopic antibody at HC1: HC2: FC1: FC2 ratio of 22:8:35:35 and 1.75mg of Salmon Sperm DNA, pTT22-AKTdd, GFP) was complexed with PEI MAX® 40,000 (PolyScience; Cat. No. 24765-1) at a 1:4 (w / w) DNA / PEI ratio. Transfections were performed with DNA / PEI complexes in 3.5L cell culture volume supplemented with IX Antibiotic / Antimycotic Solution (HyClone; Cat. No. SV30079.01) and incubated at 37°C for 24 hrs. This was followed by a temperature shift to 32°C for 6 days with 0.5 mM valproic acid and 1.0% (w / v) peptone. GFP measurements were taken 1 day post transfection to determine transfection efficiency.

[0449] Production was harvested 7 days post transfection. Cells were centrifuged, then filtered on a 0.22uM Millipore™ filter. Clarified supernatant was analyzed by small scale HPLC -Protein A for titer measurements.

[0450] Protein A Affinity Chromatography and Preparative SEC: Samples underwent Protein A chromatography and preparative SEC following the protocol described in Example 1 except that preparative SEC utilized Superdex™ 200 26 / 600 pg (Cytiva) and sample was concentrated using Vivaspin® TURBO units (Sartorius) prior to quantification based on absorbance at 280nm (A280nm).

[0451] Caliper: Purity of sample was assessed by electrophoresis under non-reducing (NR) and reducing (R) conditions following the protocol described in Example 1.

[0452] UPLC-SEC: UPLC-SEC was performed following the protocol described in Example 1.

[0453] Liquid chromatography-mass spectrometry (LC-MS): The apparent purity of the biparatopic antibody was assessed using mass spectrometry. First, the sample was diluted to 1 mg / mL, then 100 mM Tris-HCl pH 7 was added 1:1. The sample was deglycosylated with PNGaseF (Sigma) as follows: 0.1U PNGaseF / pg of antibody, overnight incubation at 37°C. The deglycosylated protein sample was analyzed by intact LC-MS using an UltiMate™ 3000 HPLC system coupled via an Ion Max electrospray source to an LTQ-Orbitrap™ XL mass spectrometer(ThermoFisher, Waltham, MA) (tuned for optimal detection of larger proteins (>50kDa), cone voltage ~40V, FT resolution 7,500, scan range 400-4000 m / z). The sample was injected onto a 2.1 x 30 mm POROS™ R2 reverse phase column (Applied Biosystems) and resolved using a 0.1 % formic acid aq / acetonitrile (degassed) linear gradient (20-90% acetonitrile). The column and solvents were heated to ~80°C to improve protein peak shape. The suitability of the LC-MS system for IgG sample analysis was evaluated using a deglycosylated IgG standard and an in-house mAb:half-antibody standard mix. For each LC-MS analysis, the mass spectra acquired across the antibody elution peak were summed and the ion envelopes were deconvoluted into a molecular weight profile using the MaxEnt 1 module of MassLynx™ (Waters, Milford, MA). Internally developed software enabled automatic assignment of different heavy and light chain combinations and determination of their apparent relative amounts from peak heights in the molecular weight profiles.

[0454] Results: The yield obtained for v39794 was ~88 mg. NR Caliper reflected the expected molecular weight for the desired species and R Caliper reflected the presence of intact heavy and light chains (Fig. 7). The UPLC-SEC profile reflected high species homogeneity, with purity of -99.9% monomeric species (Fig. 8), and the bispecific (biparatopic) purity of v39794 was determined as -89% by LC-MS (Fig. 9A-B). In Fig. 9A, the deconvoluted mass spectrum shows a predominant presence of the desired biparatopic species (“Main” in Fig. 9A) with minimal presence of half-Ab species (<2%; Fig. 9B).EXAMPLE 10: FUNCTIONAL CHARACTERIZATION OF ANTI-PTK7 BIPARATOPIC ANTIBODY - CELLULAR BINDING

[0455] The ability of the anti-PT K7 biparatopic antibody, v39794 to bind to PTK7 expressed on cells was assessed on endogenous PTK7-expressing tumor cell lines as described below. The PTK7 -targeting antibody cofetuzumab (vl9287) and the monospecific antibody, v38332 (see Example 5), were used as positive controls. Palivizumab (anti-RSV) (v21995) was used as a negative control.

[0456] Cell lines used were HCC1569 (breast carcinoma) and H446 (lung carcinoma). HCC1569 cells express endogenous PTK7 at high level and H446 cells express PTK7 at moderate level, as determined by surface protein quantification as described in Example 16. Cellular binding of thetest antibodies was assessed following the protocol described in Example 5 except after treatment with test antibody, cells were washed and stained with anti-Human IgG Fc AF647 conjugate at 4°C for 30 min.

[0457] The results are shown in Table 10.1 and plotted in Fig. 10. Anli-P'I’K? biparatopic antibody, v39794, showed comparable binding (Kd) to monospecific antibody v38332 and cofetuzumab against both HCC1569 and H446 cell lines. Biparatopic antibody v39794 showed greater binding Bmax than monospecific antibody v38332 and cofetuzumab against both HCC1569 and H446 cell lines. Negative control palivizumab showed no cellular binding, as expected.Table 10.1: Cellular Binding of Anti-PTK7 Biparatopic Antibody, v39794HCC1569 H446Sample Curve Hill Curve Hill Bmax Kd(nM) Bmax Kd(nM)Slope (h) Slope (h) v39794 15,359 0.26 6.8 9,959 0.12 1.3 v38332 9,631 0.17 1.0 6,882 0.09 0.9 Cofetuzumab 7,682 0.13 1.4 6,417 0.09 1.3 Palivizumab NB* NB NB NB NB NB* NB = no binding (apparent Kdvalue greater than the highest antibody testing concentration >150 nM) EXAMPLE 11: FUNCTIONAL CHARACTERIZATION OF ANTLPTK7 BIPARATOPIC ANTIBODY - HUMAN AND CYNOMOLGUS MONKEY PTK7 BINDING

[0458] The ability of the anti-PT K7 biparatopic antibody v38794 to bind to human and cynomolgus monkey PTK7 was assessed by flow cytometry using transfected CHO-S cells following the protocol described in Example 5. Anti-PTK7 antibody cofetuzumab (vl9287) and the monospecific antibody, v38332 (see Example 5), were included as positive controls. Palivizumab (anti-RSV) (v21995) was included as a negative control.

[0459] The results are shown in Table 11.1 and Fig. 11. On both human and cynomolgus monkey PTK7 transfected CHO-S cells, the biparatopic antibody, v39794, showed comparable binding Kd to the monospecific antibody, v38332, and to cofetuzumab. On human PTK7 transfected CHO-S cells, the biparatopic antibody, v39794, showed greater binding Bmax than cofetuzumab, yieldinga Bmax of 3-fold higher than cofetuzumab. Negative control palivizumab did not bind to PTK7 from either species, as expected.Table 11.1: Binding of Anti-PTK7 Biparatopic Antibody, v39794, to Human and Cynomolgus Monkey PTK7Sample Bmax Apparent Kd (nM)Hu PTK7 Cyno PTK7 Hu PTK7 Cyno PTK7v39794 27,534 1,820 0.87 0.10v38332 32,066 1,566 0.77 0.07Cofetuzumab 8,024 1,249 0.16 0.04Palivizumab NB* NB NB NB* NB = no binding (apparent Kdvalue greater than the highest antibody testing concentration (>200 nM)) EXAMPLE 12: BINDING AFFINITY ASSESSMENT OF ANTI-PTK7 BIPARATOPIC ANTIBODY

[0460] Surface plasmon resonance (SPR) analysis was performed to characterize binding affinity of the anti-PTK7 biparatopic antibody (v39794) to human and cynomolgus monkey PTK7 antigens. Cofetuzumab (vl9287) was used as a positive control. A Cytiva Biacore™ T200 instrument (Cytiva, Marlborough, MA) was used for the characterization following the protocol described in Example 8 except the anti-Fc antibody was injected into flow cells 1 to 2 only, and test article at a concentration of 4 pg / mL was injected into flow cell 2 as a binding ligand. In addition, to avoid possible surface avidity, test articles were captured by immobilized anti-Fc antibody at a low ligand density around 50 RU.

[0461] The results of the SPR binding analysis are shown in Table 12.1. The rate of complex formation is represented as the association constant kon, and the rate of complex dissociation is represented by koff. The affinity of the analyte for the ligand (the equilibrium dissociation constant, KD) is calculated from the kinetic association and dissociation rates koff / kon). The biparatopic variant v39794 showed similar affinities (KD) for both cynomolgus and human PTK7 antigens, and exhibited comparable KD values to cofetuzumab (v 19287) at a nanomolar scale.Table 12.1: SPR Binding Analysis of Anti-PTK7 Biparatopic Antibody to Human and Cynomolgus Monkey PTK7Common AntigenTest Article kon(1 / Ms) koff (1 / s) KD (nM) Name Species13G11 x Human 2.38E+05 2.53E-04 1.07 v3979414D08 Cynomolgus 2.14E+05 2.93E-04 1.37Human 1.98E+05 6.15E-04 3.11 V19287 CofetuzumabCynomolgus 1.58E+05 4.44E-04 2.80EXAMPLE 13: FUNCTIONAL CHARACTERIZATION OF ANTLPTK7 BIPARATOPIC ANTIBODY - INTERNALIZATION

[0462] The PTK7-mediated internalization capability of the anti-PTK7 biparatopic antibody, v39794, in PTK7-expressing cell lines was determined by flow cytometry following the protocol described in Example 6. The PTK7-targeting antibody cofetuzumab (vl9287) and the anti-PTK7 monospecific antibody, v38332 (see Example 5), were used as positive controls. The anti-RSV antibody palivizumab was used as a negative control.

[0463] Cell lines used were HCC1569 (breast carcinoma) and H446 (lung carcinoma). HCC1569 express endogenous PTK7 at high level and H446 express PTK7 at moderate level, as determined by surface protein quantification in as described Example 16.

[0464] Results'. The results for the 10 nM test concentration are tabulated in Table 13.1 and shown in Fig. 12. Positive control cofetuzumab (vl9287) showed internalization in HCC1569 and H446, as expected. Anti-PTK7 biparatopic antibody, v39794, showed increased internalization compared to palivizumab negative control in both cell lines evaluated. Following a 4-hour incubation in HCC1569 and H446, v39794 showed greater internalized fluorescence than the anti-PTK7 monospecific antibody, v38332, and benchmark cofetuzumab, yielding 2-3-fold increase in internalized fluorescence over cofetuzumab at 10 nM treatment.Table 13.1: Internalization of Anti-PTK7 Biparatopic Antibody (lOnM Treatment) Internalized FluorescenceAntibody (Fold-Over Palivizumab)HCC1569 H446v39794 41.3 30.8v38332 17.2 10.8Cofetuzumab 18.6 10.8Palivizumab 1.0 1.0EXAMPLE 14: FUNCTIONAL CHARACTERIZATION OF ANTLPTK7 BIPARATOPIC ANTIBODY - SPHEROID PENETRATION

[0465] The ability of the anti-PTK7 biparatopic antibody v39794 and the anti-PTK7 monospecific antibodies v39115 and v39133 to penetrate PTK7-expressing cell line spheroids was assessed as described below. The PTK7 -targeting antibody cofetuzumab (v 19287) and the high-affinity monospecific anti-PTK7 antibody, v41357, were included as positive controls. Variant v41357 is identical to v38332 (see Example 5) but includes a lysine residue at the C-terminus of the heavy chain. Palivizumab (anti-RSV, v21995) was included as a negative control.

[0466] The cells lines used were H446 and HCT116. Both these cell lines express endogenous PTK7 at moderate levels, as determined by surface protein quantification as described in Example 16.

[0467] Briefly, antibodies were fluorescently labeled by coupling to a Fab fragment AF488 conjugate targeting human IgG Fc (Jackson ImmunoResearch Inc, West Grove, PA; Cat. No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No.10010-023), for 24 hours at 4°C. Spheroids were formed by seeding H446 or HCT116 cells (10,000 cells / well) into Corning® 96-well round bottom ultra-low attachment surface spheroid microplates (Millipore Sigma, Oakville, ON, Canada; Cat. No. CES4520) using complete growth medium (RPMI medium, ATCC modification (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. A1049101) or McCoy’s 5A medium, ATCC modification (Thermo Fisher Scientific, Waltham, MA; Cat. No. 16600082), respectively, supplemented with 10% fetal bovine serum).Plates were centrifuged at 200 x g for 2 minutes and incubated for 2 days under standard culturing conditions (37°C / 5% CO2) to allow for spheroid formation. After incubation, coupled antibodies were added to spheroids at 50 nM and incubated under standard culturing conditions for 24 to 48 hours. Following incubation, spheroids were washed to remove unbound antibody by adding 100 µL fresh complete growth medium and removing 100 µL medium from the well, for a total of three washes. Cell nuclei were stained with Hoechst 33342 dye (Thermo Fisher Scientific, Waltham, MA; Cat. No. 62249) at 10 pM and excess and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A- 11094) at 100 nM for 2 hours at 37°C / 5% CO2.

[0468] Imaging was performed using an Operetta™ CLS™ high content analysis system (Perkin Elmer, Waltham, MA). Image analysis was performed using Harmony® 4.5 software (Perkin Elmer, Waltham, MA). 2-dimensional analysis was performed on the central slice of each spheroid by dividing the spheroid object into ten subregions of concentric bands, each representing 10% area of the total spheroid object. AF488 mean fluorescence intensity (MFI) within each subregion band was quantified, corrected by subtracting the inner 10% AF488 MFI of v21995 -treated spheroids. The AF488 MFI of two adjacent regions were summed to generate five “layers” and plotted using GraphPad Prism Software Version 10 (GraphPad, Fa Jolla, CA).

[0469] The results are shown in Tables 14.1 and 14.2 and in Fig. 13. Following 24-hour incubation of H446 and HCT116 spheroids with 50 nM fluorescently-labeled test antibodies, the biparatopic antibody v39794 demonstrated effective spheroid binding and penetration, with high MFI detectable into the center layers and peak MFI at 426 and 450 for H446 and HCT116 spheroids, respectively. In comparison to the biparatopic antibody, PTK7 -targeting monospecific antibodies (including positive control cofetuzumab) demonstrated lesser spheroid binding and penetration but still achieved detectable MFI in the center layers. Monospecific antibodies showed comparable spheroid penetration capability to each other, with peak MFI ranging from 184-271 and 175-200 for H446 and HCT116 spheroids, respectively. Similarly, following 48-hour incubation of H446 and HCT116 spheroids with 50 nM fluorescently-labeled test antibodies, the biparatopic antibody v39794 demonstrated greatest spheroid binding and penetration, with high MFI detectable into the center layers and peak MFI at 563 and 502 for H446 and HCT116 spheroids, respectively, while monospecific antibodies (including positive control cofetuzumab)demonstrated lesser spheroid binding and penetration with peak MFI ranging from 214-312 and 225-283 for H446 and HCT116 spheroids, respectively. At both time points, negative control antibody, palivizumab, showed minimal MFI at all spheroid layers, indicative of minimal spheroid binding and penetration, as expected.Table 14.1: Penetration of Anti-PTK7 Biparatopic and Monospecific Antibodies into H446 SpheroidsSpheroid Sample (50 Time Layer 1 Layer 2 Layer 3 Layer 4Center nM) (h)Average Corrected AF488 Mean Fluorescence Intensity 24 426 418 381 372 364 v3979448 315 563 484 458 472 24 211 190 165 161 156 v4135748 249 252 220 198 196 24 271 261 232 216 225 v3913348 301 312 268 246 239 24 241 230 208 199 194 v3911548 189 255 207 189 188 24 184 179 158 142 141 Cofetuzumab48 214 203 169 147 142 24 63 42 29 8 1 Palivizumab48 24 19 2 -9 -5Distribution (%)24 22% 21% 19% 19% 19% v3979448 14% 25% 21% 20% 21% 24 24% 21% 19% 18% 18% v4135748 22% 23% 20% 18% 18% 24 23% 22% 19% 18% 19% v3913348 22% 23% 20% 18% 18% 24 22% 21% 19% 19% 18% v3911548 18% 25% 20% 18% 18% Cofetuzumab 24 23% 22% 20% 18% 18%48 24% 23% 19% 17% 16% 24 44% 30% 20% 6% 1% Palivizumab48 78% 62% 8% -30% -17%Table 14.2: Penetration of Anti-PTK7 Biparatopic and Monospecific Antibodies into HCT116 SpheroidsSpheroid Sample (50 Time Layer 1 Layer 2 Layer 3 Layer 4Center nM) (h)Average Corrected AF488 Mean Fluorescence Intensity 24 286 450 416 376 344 v3979448 266 502 488 444 421 24 126 197 200 177 159 v4135748 171 283 262 231 213 24 117 173 175 138 123 v3913348 149 230 237 214 196 24 146 188 191 176 165 v3911548 142 225 202 177 167 24 144 181 168 147 136 Cofetuzumab48 146 226 185 154 145 24 56 23 6 -1 -2 Palivizumab48 49 21 6 -1 -1Distribution (%)24 15% 24% 22% 20% 18% v3979448 13% 24% 23% 21% 20% 24 15% 23% 23% 21% 18% v4135748 15% 24% 23% 20% 18% 24 16% 24% 24% 19% 17% v3913348 14% 22% 23% 21% 19% 24 17% 22% 22% 20% 19% v3911548 16% 25% 22% 19% 18% 24 19% 23% 22% 19% 18% Cofetuzumab48 17% 26% 22% 18% 17%24 69% 28% 7% -2% -3% Palivizumab48 66% 28% 8% -1% -1%EXAMPLE 15: PREPARATION AND CHARACTERIZATION OF ANTIBODY-DRUG CONJUGATES COMPRISING ANTI-PTK7 BIPARATOPIC ANTIBODY

[0470] Antibody-drug conjugates (ADCs) comprising the anti-PTK7 biparatopic antibody, v39794, linked by thiol conjugation to a topoisomerase 1 inhibitor or microtubule inhibitor were prepared and the ADCs characterized as described below. The structures of the drug-linkers, DL- 1 (topoisomerase 1 inhibitor) and DL-2 (microtubule inhibitor) employed are shown below. DL-1:Conjugation

[0471] The antibody (1-10 mg / mL in phosphate buffered saline, pH 7.4) was reduced with tris(2-carboxyethyl)phosphine (TCEP) (1-10 mM in dH₂O) (12 eq.) in the presence of 1 mMdiethylenetriaminepentaacetic acid (DTP A). The solution was mixed thoroughly and incubated at 37 °C for 180 min before cooling on ice. In some instances, the reduced antibody solution was then further buffer exchanged into 10 mM sodium acetate buffer, pH 4.5 by passage over a Zeba™ Spin Desalting Column (Thermo Scientific; Cat. No. A57765). To the reduced protein solution was added the maleimide functionalized drug-linker (10 mM in DMSO) (8-16 eq.). In some instances, dimethyl sulfoxide (DMSO) (10 percent v / v%) was added to the reduced protein solution prior to the addition of drug-linker. The conjugation reaction was immediately mixed thoroughly by pipetting and conjugation was allowed to proceed at room temperature for 60 to 120 min while mixing. In some instances where the ADC was produced at >5 mg scale, the conjugation reaction was followed by quenching of excess drug-linker with 10 mM aqueous stock of N-acetyl cysteine (8-12 eq.) for 30 to 60 min. Mixing was achieved by rotating the reaction tubes on a tube revolver (Thermo Scientific; Cat. No. 88881001).

[0472] Once complete, purification of the ADC from small molecules was performed by passage over a Zeba™ Spin Desalting Column (Thermo Scientific; Cat. No. A57765) into 50 mM sodium acetate pH 5.5 or PBS pH 7.4. Where the ADC was produced at >5 mg scale, purification was performed on an AKTApure™ FPLC operated by Unicorn™ 7 software (Cytiva; Cat. No. 29018224). A pre-packed HiPrep™ 26 / 10 desalting column (Cytiva; Cat. No. 17508701) with Sephadex G-25 resin was used per manufacturer’s standard and the protein was eluted with 50 mM sodium acetate pH 5.5 or PBS pH 7.4. Selected fractions based on their UV absorbance and area were pooled for analysis. The pooled and purified conjugates were sterile filtered (0.22 mm), stored at 4 °C and analyzed for total protein content by spectrophotometric protein quantification at 280 nm or bicinchoninic acid assay (Pierce micro-BCA protocol; Cat. No.23225).

[0473] Characterization of the ADCs was performed by HPLC -hydrophobic interaction chromatography (HIC), HPLC-size exclusion chromatography (SEC), capillary electrophoresis (CE)-SDS and reverse phase-HPLC-mass spectrometry (RP-HPLC-MS). The average DAR and drug distribution was derived from interpretation of HIC and / or LC-MS data. Endotoxin levels were assessed using Endosafe® LAL test cartridges (Charles River; Cat No. PTS20005F), with a sensitivity of 0.005 EU / mL on an Endosafe® nexgen-PTS™ testing system. Residual free compound and drug-linker levels were assessed by RP-UPLC-MS or RP-UPLC-fluorescencedetection (FLD), with a threshold set at 1% ((free compound + drug-linker) / (conjugated druglinker)).PAR Determination by HIC

[0474] The average DAR by HIC was assessed as described in Antibody Drug Conjugates, Methods in Molecular Biology, 2013, vol. 1045, pp. 275-284. L. Ducry, Ed. The experiments were performed on an Agilent Infinity II 1290 HPLC (Agilent Technologies, Santa Clara, CA) using a TSKgel® Butyl-NPR column (2.5pm, 4.6 x 35mm; TOSOH Bioscience GmbH, 20 Griesheim, Germany) pre-equilibrated with 5 column volumes of Buffer A (1.5 M (NH₄)₂SO₄, 25 mM NaH₂PO₄, pH = 6.95) and at room temperature. Typically, 10-30 µg of sample at 2-3 mg / mL concentration were loaded on the column with 95% Mobile Phase A and 5% Mobile Phase B (75% 25 mM NaH₂PO₄ plus 25% isopropanol, pH 6.95) and run for 15 mins at 0.5 mL / min using the gradient shown in Table 15.1. HIC chromatograms were integrated using appropriate parameters that provided complete, baseline -to-baseline integration of each peak, followed by integration of each peak showing reasonable separation. As a reference, unconjugated naked antibody was run on the same gradient to obtain the HIC retention time for DAR = 0 species.Table 15.1: HPLC-HIC GradientTime (min) % Buffer A % Buffer B0 95 512 5 9512.1 95 515 95 5DAR Determination by RP-HPLC-MS

[0475] ADC samples were deglycosylated using Endo S for 1 hour at RT, followed by reduction with TCEP at a final concentration of 500µM for 1 hour at RT. Samples were injected onto an Agilent 1290 Infinity II LC coupled with an Agilent 6545 Quadrupole Time of Flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA). Protein species were separated using a BioSuite™ pPhenyl RPC column (1000A, 10 pM, 75 x 2.0 mm; Waters, Milford, MA) maintained at 70 °C, at a flow rate of 0.3 mL / min with the gradient shown in Table 15.2. Mobilephase A: 0.1% formic acid (FA), 0.025% trifluoroacetic acid (TFA) and 10% isopropyl alcohol (IP A) in water. Mobile phase B: 0.1% FA and 10% IPA in acetonitrile (ACN).Table 15.2: RP-HPLC-MS GradientTime (min) % Buffer A % Buffer B0 90 1020 73 2722 10 9022.5 1 9924 1 99

[0476] The MS source conditions are shown in Table 15.3 and the acquisition parameters were as follows: Mode'. MS; Mass Range: 500 to 7000 m / z; Acquisition Rate: 1 spectra / s and 1000 ms / spectrum, 3352 transients / spectrum.Table 15.3: MS Source ConditionsGas Temp: 300°C VCap: 5000VDrying Gas: 13 l / min Nozzle Voltage: 2000VNebulizer: 45 psig Fragmentor: 170 VSheath Gas Temp: 400°C Skimmer: 65VSheath Gas Flow: 12 l / min Oct RF Vpp: 750V

[0477] Protein Metrics Byos® (Protein Metrics Inc., Cupertino, CA) was employed for deconvolution and qualitative data analysis. Deconvolution parameters were as follows: Peak integration, MS deconvolution and mass assignments were performed in Protein Metrics Byos® v4.6 using a deconvolution window of 20000-160000 Da with an m / z range of 1000-6000. For all time points, the highest intensity deconvoluted mass was assigned as the reference mass. The reference mass was defined as the average mass of variant with two 2-acetamido-2-deoxy-beta-D-glucopyranose-(l-4)-[alpha-L-fucopyranose-(l-6)] stubs arising from EndoS activity on N-glycans, 16 disulfide bonds and the formation of pyroglutamic acid, if applicable, at the N-terminus. Mass assignments had a mass tolerance of ±10 Da. Other adducts assigned based on mass shifts relative to the reference mass were: variant reference mass with a phosphoric acidadduct, variant reference mass with the loss of one fucose unit, variant reference mass with the addition of a hexose unit. A mannose-5 adduct relative to the reference mass of variant without 2-acetamido-2-deoxy-beta-D-glucopyranose-(l-4)-[alpha-L-fucopyranose-(l-6)] was also identified.

[0478] Average DAR was the calculated from the deconvoluted spectrum using the following equation:Z nDruq load, ■ peak intensity,- T -otal 7“ p —ea 7k - intensity ~SEC-HPLC Analysis of ADCs

[0479] Analytical SEC was performed using an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) with an Advance Bio SEC column (300 A, 2.7 pm, 7.8 x 300 mm; Agilent Technologies) equilibrated with 5 column volumes of Buffer (150 mM Na₂PO₄, pH 6.95) at room temperature. Typically, 10 pg of sample at 1-3 mg / mL concentration was eluted isostatically for 12 mins at 1 mL / min and absorbance monitored at A280. Chromatograms were integrated to provide complete, baseline -to-baseline integration of each peak, with reasonably placed separation between partially resolved peaks. The peak corresponding to the major component for IgG (approximate retention time 6.5 min) was reported as the monomer based on the SEC profile of unmodified antibodies. Any peak occurring prior to 6.5 min was designated as high molecular weight species (HMWS), and any peak occurring after 6.5 min was designated as low molecular weight species (LMWS), excluding solvent peaks (over 9.0 min).CE-SDS Analysis of ADCs

[0480] Initially, all samples were diluted to 1 mg / mL before preparing the samples in a 96-well 10 PCR plate following manufacturer’s protocol (Protein Express Assay LabChip™; PerkinElmer, Inc., Waltham, MA). Briefly, 2 mg of ADC was mixed with 7 pL Protein Express buffer in the presence (reducing) or absence (non-reducing) of 34 mM dithiothreitol (DTT), followed by heat denaturation at 95°C for 5 minutes. Samples were then diluted in dH2O at a 9:44 ratio before data acquisition using the HT Protein Express Chip (PerkinElmer Inc.; Cat No. 760499) and the HTProtein Express 200 Assay Setting. After each CE-SDS run, the electropherogram and corresponding gel were analyzed using LabChip™ Reviewer (PerkinElmer, Inc., Waltham, MA). The percent purity was determined by the sum of partially or fully reduced species.Results

[0481] The biophysical properties of the ADCs are summarized in Table 15.4.Table 15.4: Biophysical Properties of v39794 ADCsReported DAR Monomer % Free% Purity Target % Drug Endotoxin ADC by CE- DAR HPLC- (HPLC- (mol% / (EU / mg)LC-MS SDS HIC SEC) DAR)v39794- 8 7.5 8.0 100 0.0 0.2 99.3 DL-1v39794- 4 3.8 4.0 97.8 0.0 0.03 100 DL-1v39794- 4 3.6 4.7 95.7 1.1 ND* ND DE-2* ND = not determinedEXAMPLE 16: PTK7 QUANTIFICATION BY FLOW CYTOMETRY

[0482] The average number of PTK7 proteins per cell for the cancer cell lines listed in Table 16.1 was determined by flow cytometry as described below. Quantum™ Simply Cellular® (QSC) anti-Human IgG Beads (Bangs Laboratories, Cat. No. 816C) containing 5 bead populations were used: 1 blank and 4 with increasing levels of Fc-specific capture antibody.

[0483] Briefly, cells were seeded at 50,000 cells / well and 20 pL of each QSC anli-I luinan IgG Bead population was added to 96-well plates. Cells and bead populations 1-4 were then pelleted by centrifugation, washed, and resuspended with 100 µL / well of cofetuzumab (v19287), or negative control anti-RSV antibody (palivizumab; v21995), conjugated to Alexa Fluor® 647 at saturating concentration (15 µg / mL). Blank bead population was resuspended in blank buffer only (PBS pH 7.4 and 2% FBS). The 96-well plate was incubated for 30 minutes on ice, in the dark. Following incubation, cell fluorescence was analyzed by flow cytometry with BD LSR Fortessa™(BD Biosciences, Franklin Lakes, NJ, USA). Each treatment sample captured at least a minimum of 1,000 events.

[0484] For data analysis and PTK7 / cell calculation, AF647 mean fluorescence intensity (MFI) values for all bead populations were plotted against relevant antibody binding capacity (ABC) values using Bangs Laboratories QuickCal® v 2.3 calibration line template for QSC® anti-Human IgG Lot # 14490. The AF647 MFI values for all cell samples stained with V19287-AF647 were applied to their respective calibration line by linear regression to obtain ABC values for all cell samples. Surface PTK7 expression for cell lines was calculated based on a monovalent binding model and therefore equivalent to the background-subtracted ABC (SABC). The AF647 MFI values of v21995-AF647-stained cells from each of the respective cell lines were subtracted from the MFI values of v19287-AF647-stained cells for determination of SABC. Average PTK7 / cell values are provided in Table 16.1.

[0485] A range of PTK7 expression, ranging from 0 to 1,115,708 PTK7 / cell, was determined in the six cancer cell lines evaluated. A cell line with over 700,000 PTK7 / cell is considered as a high PTK7-expressing cell line. A cell line expressing between 200,000 and 700,000 PTK7 / cell is considered a moderate PTK7-expressing cell line. A cell line with less than 200,000 PTK7 / cell is considered a low PTK7-expressing cell line. SABC values that were negative were assigned a value of 0 in the averaging calculation for reporting surface average PTK7 / cell and a cell line with 0 PTK7 / cell is considered as a negative cell line.Table 16.1: Quantification of PTK7 Protein on Cancer Cell LinesAverage PTK7 Cell Line Source OriginPTK7 / cell' Expression ATCC® CRL-2330™ 1,115,708 ± HCC1569 Breast Carcinoma High Manassas, VA 88,804 ATCC® HTB-123™DU4475 Breast Carcinoma 784,685 High Manassas, VAATCC® HTB-171™ 650,845 ± H446 Lung Carcinoma Moderate Manassas, VA 119,599 ATCC® CCL-247™ Colorectal 279,093 ± HCT116 Moderate Manassas, VA Carcinoma 28,133Average PTK7 Cell Line Source OriginPTK7 / cell1Expression ATCC® CRL- 11730™ OvarianTOV-21G 74,665 Low Manassas, VA AdenocarcinomaATCC® HTB-30™ BreastSK-BR-3 3,965 ± 1,158 Low Manassas, VA AdenocarcinomaATCC® CRL- 1682™ PancreaticAsPC-1 02Negative Manassas, VA Adenocarcinoma1Average of 1 to 6 independent biological experiments2SABC values that were negative were assigned a value of 0 in the averaging calculation for reporting surface average PTK7 / cellEXAMPLE 17: IN VITRO 2D MONOLAYER AND 3D SPHEROID CYTOTOXICITY OF ANTI-PTK7 ANTIBODY-DRUG CONJUGATES

[0486] The cell growth inhibition (cytotoxicity) capabilities of the ADC comprising the biparatopic anti-PTK7 antibody v38794 conjugated to DL-1 at a DAR of 8 (see Example 15), and ADCs comprising the monospecific anti-PTK7 antibodies v41357, v38115 or v38133, each conjugated to DL-1 at a DAR of 8 (prepared following the protocol described in Example 15), was assessed in a panel of PTK7-expressing cell lines in 2D monolayer and 3D spheroid formats as described below. An ADC comprising cofetuzumab conjugated to DL-1 at a DAR of 8 was used as positive control, and the anti-RSV antibody palivizumab, conjugated to DL-1 at a DAR of 8, was included as a non-targeted control.

[0487] Cell lines used were H446 (lung carcinoma), DU4475 (breast carcinoma) and AsPC-1 (pancreatic adenocarcinoma) for 2D cytotoxicity assessment and the same cell lines plus HCC1569 (breast carcinoma) for 3D cytotoxicity assessment. HCC1569 cells and H446 cells express endogenous PTK7 at high levels; DU4475 cells express endogenous PTK7 at moderate levels and AsPC-1 are characterized as PTK7-negative, as determined by surface protein quantification as described in Example 16.

[0488] Cells were maintained under standard culture conditions (37°C / 5% CO2) until assay setup: all cell lines were cultured in complete growth medium (RPMI 1640, ATCC modification(Thermo Fisher Scientific, Waltham, MA, Cat. No. A1049101) supplemented with 10% fetal bovine serum).

[0489] Briefly, for spheroid generation, cells were seeded into 384-well round bottom ultra-low attachment (ULA) microtiter plates (Corning, Darmstadt, Germany; Cat. No. CLS3830) and incubated for 2 days at 37°C / 5% CO2 prior to test article addition. For monolayer format, cells were seeded into 384-well tissue culture -treated flat bottom plates (Thermo Fisher Scientific, Toronto, CA; Cat No. 164610) and treated with test article on the same day. Test articles were serially diluted in complete growth medium to create a 9-point dose -response curve, including growth medium-only blank controls, and added to cells in each of monolayer and spheroid formats in the microtiter plates. Monolayer and spheroid plates were incubated at 37°C / 5% CO2 for 4 and 6 days, respectively. After incubation, cell viability was measured by addition of ATP metabolism detection reagents, CellTiter-Glo® (Promega Corporation, Madison, WI; Cat. No. G7573) or CellTiter-Glo® 3D (Promega Corporation; Cat. No. G9683), to monolayer or spheroid plates, respectively. Resulting luminescence signal was measured using the Synergy™ Hl plate reader (BioTek Instruments, Winooski, VT). Percent cytotoxicity values were calculated based on blank wells (no test article added) and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA). EC₅₀ values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 9.Results

[0490] 2D cytotoxicity results are shown in Table 17.1 and plotted in Fig. 14A-C. ADCs of v41357, v38115, v38133, v38794 and cofetuzumab conjugated to DL-1 demonstrated dosedependent cytotoxicity in PTK7-expressing H446 and DU4475 monolayers compared to the nontargeting control ADC (see Fig. 14A-B). ADCs of v41357 and cofetuzumab yielded incomplete curves within the concentration range tested in H446 and DU4475 monolayers and the ADC of v38115 yielded incomplete curves within the concentration range tested in H446 monolayer. In contrast, no targeted killing was observed in PTK7-negative AsPC-1 cells (see Fig. 14C).

[0491] 3D cytotoxicity results are shown in Table 17.2 and plotted in Fig. 15A-D. ADCs of v41357, v38115, v38133, v38794 and cofetuzumab conjugated to DL-1 demonstrated dose-dependent cytotoxicity in PTK7-expressing HCC1569, H446, and DU4475 spheroids compared to the non-targeting control ADC (see Fig. 15A-C). In contrast, no targeted killing was observed in PTK7-negative AsPC-1 cells (see Fig. 15D).Table 17.1: In Vitro Cytotoxicity of Anti-PTK7 ADCs in 2D Monolayer FormatEC50 (nM)ADC DARH446 DU4475 AsPC-1V41357-DL-1 7.7 IC* IC >200V38115-DL-1 8.0 IC 0.02 >200V38133-DL-1 7.7 0.02 0.01 >200V38794-DL-1 7.8 0.01 0.01 >200Cofetuzumab-DL- 1 7.8 IC IC >200Palivizumab-DL- 1 8.0 >200 32.98 >200*IC = Incomplete curveTable 17.2: In Vitro Cytotoxicity of Anti-PTK7 ADCs in 3D Spheroid FormatEC50 (nM)ADC DAR HCC1569 H446 DU4475 AsPC-1V41357-DL-1 7.7 0.08 0.02 0.04 >200V38115-DL-1 8.0 0.31 0.14 0.16 >200V38133-DL-1 7.7 0.11 0.06 0.10 >200V38794-DL-1 7.8 0.11 0.05 0.09 >200 Cofetuzumab-DL- 1 7.8 0.28 0.09 0.10 >200 Palivizumab-DL- 1 8.0 42.25 31.33 >200 >200EXAMPLE 18: BYSTANDER ACTIVITY OF ANTI-PTK7 ANTIBODY-DRUG CONJUGATES

[0492] The bystander killing effect of ADCs comprising the biparatopic anti-PTK7 antibody v38794 conjugated to DL-1 at DAR 8 (see Example 15) or the monospecific anti-PTK7 antibody v41357 conjugated to DL-1 at DAR 8 (prepared following the protocol described in Example 15)on cancer cells was assessed as described below. All test articles were assessed at 1 nM treatment. Palivizumab (v21995)-DL-1 DAR 8 was included as a negative control.

[0493] Cell lines used were H446 (lung carcinoma) and SK-BR-3 (breast adenocarcinoma). H446 cells express endogenous PTK7 at high levels and SK-BR-3 cells express PTK7 at low levels, as determined by surface protein quantification as described in Example 16.

[0494] Cells were maintained under standard culture conditions (37°C / 5% CO2) until assay setup; H446 cells were cultured RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA; Cat. No. A1049101) and SK-BR-3 cells were cultured in McCoy's 5 A, ATCC modification (Thermo Fisher Scientific; Cat. No. 16600082), supplemented with 10% fetal bovine serum.

[0495] Briefly, H446 and SK-BR-3 cells were seeded either as monocultures or co-cultures in a 48-well tissue culture-treated flat bottom plates (Greiner CELLSTAR, Ottawa, CA; Cat. No.677180) at 20,000 cells and 10,000 cells per well, respectively. Cells were then treated with ADCs at a final 1 nM concentration and incubated for 4 days under standard culturing conditions (37°C / 5% CO2). Following incubation, cells were dissociated, washed, and stained using a viability dye, YO-PRO®-1 (Thermo Fisher Scientific, Waltham, MA; Cat. No. Y3603), and the anti-PTK7 antibody, cofetuzumab (vl9287), conjugated to Alexa Fluor® 647, for 20 minutes at 4°C. After incubation, cells were washed in FACS buffer, resuspended in 60 pF FACS buffer per well, and 35 pF per well were analyzed on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ). Dead cells were excluded by gating on YO-PRO®-1 staining. The number of H446 and SK-BR-3 cells were determined by the number of events in the Alexa Fluor® 647 positive (PTK7-positive) and Alexa Fluor® 647 negative (PTK7-negative) gates, respectively. Percent viability was calculated as the number of cells in each treatment condition divided by the number of cells in the no-treatment condition.

[0496] The results are shown in Table 18.1 and Fig. 16A-B. Bystander activity was calculated as the difference in viability of PTK7-low SK-BR-3 cells when treated in co-culture with PTK7-high H446 cells compared to treatment in monoculture. Anti-PTK7 biparatopic ADC v39794-DL-1 DAR 8 and anti-PTK7 monospecific ADC v41357-DL-1 DAR 8 demonstrated bystander killing capability, with the biparatopic ADC v39794-DL-1 showing greater bystander activity than themonospecific ADC v41357-DL-l. Negative control palivizumab-DL-1 displayed minimal killing in SK-BR-3 in monoculture and in co-culture with H446 cells, indicating negligible bystander activity, as expected.Table 18.1: Bystander Activity of Anti-PTK7 ADCs against PTK7-low SK-BR-3 Cell Line Average % Viability SK- Average % Viability H446Sample %DAR BR-3 Cells Cells Bystander (I nM)Monoculture Co-culture Monoculture Co-culture activity v39794-DL-l 8 105.2 % 47.2 % 10.3 % 6.7 % 32.5 % v41357- DL-1 8 102.8 % 60.8 % 20.1 % 15.3 % 20.3 % v21995- DL-1 8 106.2 % 98.5 % 112.2 % 108.3 % -0.2 %EXAMPLE 19: PHARMACOKINETICS OF ANTI-PTK7 ANTIBODY-DRUG CONJUGATES IN MOUSE CDX STUDIES

[0497] The pharmacokinetics of ADCs comprising the biparatopic anti-PTK7 antibody v38794 conjugated to DL-1 at DAR 8 (see Example 15) or the monospecific anti-PTK7 antibody v38332 conjugated to DL-1 at DAR 8 (prepared following the protocol described in Example 15) were assessed in a mouse cell line-derived xenograft (CDX) study as described below. Cofetuzumab pelidotin (cofetuzumab conjugated to an auristatin analogue, Aur0101, at DAR 4) was included as a benchmark control, and palivizumab-DL-1 DAR 8 was included as a non-targeting control.

[0498] The anti-PTK7 DAR 8 ADCs and palivizumab-DL-1 DAR 8 ADC were administered once at 6 mg / kg to BALB / c Nude mice bearing established NCI-H292 lung CDX tumors by intravenous injection. Cofetuzumab pelidotin was administered once at 2.5 mg / kg dose in the same CDX model. For each test article, blood was collected from n=3 animals by retro-orbital bleed at 1 hour, and 1, 3, 7, 14 and 21 days post-dose. Blood was processed to serum and stored frozen at -80°C in screw-cap microfuge tubes prior to pharmacokinetic analysis.

[0499] Test article Total Antibody (TAb) concentrations were measured in mouse serum by sandwich ELISA with a goat anti-human IgGl Fc capture antibody (Jackson Immuno Research Laboratories, West Grove PA; Cat. No. 109-005-098) and an HRP-conjugated goat anti-human IgGl Fab detection antibody (Jackson Immuno Research Laboratories; Cat. No. 109-035-097) fortotal IgG levels. Absorbance at 450 nM was measured using a Synergy™ H1 Hybrid Multi-Mode Plate Reader (Agilent BioTek, Santa Clara, CA). Pharmacokinetics parameters were calculated from non-compartmental analysis using Phoenix WinNonlin™ software (Certara, Princeton, NJ).

[0500] The serum concentration results are shown in Fig. 17. Analysis in this model demonstrated that total antibody (TAb) PK of the anti-PTK7 biparatopic ADC (v39794-DL-1) was comparable to the anti-PTK7 monospecific ADC (v38332-DL-l) and palivizumab-DL-1 ADC, all with typical antibody-like prolonged exposures.

[0501] Pharmacokinetic parameters are summarized in Tables 19.1 and 19.2. Elimination halflife of anti-PTK7 biparatopic ADC (v39794-DL-l), anti-PTK7 monospecific ADC (v38332-DL-1) and palivizumab-DL-1 ADC were 4.8 days, 6.7 days and 6.3 days respectively in the NCI-H292 model (Table 19.1). Clearance of v39794-DL-1, anti-PTK7 monospecific ADC (v38332-DL-1) and palivizumab-DL-1 ADC were 10.1 mL / day / kg, 10.8 mL / day / kg and 8.9 mL / day / kg respectively in the NCI-H292 model (Table 19.2). Cofetuzumab pelidotin yielded an elimination half-life of 2.5 days (Table 19.1).Table 19.1: Pharmacokinetic Parameters in NCI-H292 CDX ModelADC Dose (mg / kg) T1 / 2(day) Cmax(ug / mL) Tmax(day) V38332-DL-1 DAR8 6 6.7 106.3 0.04 V39794-DL-1 DAR8 6 4.8 135.3 0.04 Palivizumab-DL-1 DAR8 6 6.3 205.5 0.04 Cofetuzumab pelidotin 2.5 5.4 37.3 0.04Table 19.2: Pharmacokinetic Parameters in NCI-H292 CDX ModelCLDose VAD Clast Tlast AUClast zC (mg / kg) (mL / day (ug / mL) (day) (day*ug / mL) (mL / kg) / kg) V38332-DL-1 DAR8 6 5.1 21 508.4 103.7 10.8 V39794-DL-1 DAR8 6 4.8 21 562.7 70.0 10.1 Palivizumab-DL- 16 7.4 21 607.0 80.8 8.9 DAR8CL DoseADC Clast Tlast AUClast Vz(mL / day (mg / kg) (ug / mL) (day) (day*ug / mL) (mL / kg) / kg) Cofetuzumab2.5 1.7 21 233.6 79.5 10.1 pelidotinEXAMPLE 20: IN VIVO EFFICACY OF ANTI-PTK7 BIPARATOPIC ANTIBODYDRUG CONJUGATE IN A CELL LINE-DERIVED XENOGRAFT (CDX) MODEL #1

[0502] The in vivo efficacy of the ADC comprising the anti-PTK7 biparatopic antibody v39794 conjugated to DL-1 at DAR 8 (see Example 15) was tested in the PTK7-expressing NCI-H292 cell line-derived xenograft (CDX) model of human lung cancer as described below. PTK7 expression was confirmed by a research-grade IHC protocol. The anti -tumor activity of v39794-DL-l DAR 8 was compared to an ADC comprising the anti-PTK7 monospecific ADC (v38332-DL-l DAR 8; prepared following the protocol described in Example 15), a non-targeting isotype control (palivizumab) ADC conjugated to DL-1 at DAR 8, and to cofetuzumab pelidotin. The dose selected for administration of the anti-PTK7 biparatopic and monospecific ADCs were based on clinically relevant doses for known ADCs comprising topoisomerase 1 inhibitor payloads. The dose selected for administration of cofetuzumab pelidotin was based on the published clinically relevant dose.

[0503] NCI-H292 human lung cancer cells (5 x 106cells per mouse) were suspended in 0.1 mL PBS and injected subcutaneously into the right front flank region of 6-8-week-old female SCID Beige mice. Tumors were measured using a caliper and tumor volume (V, mm3) was determined by the formula V = (L x W x W) / 2, where L and W are the length and width of the tumour, respectively. When tumors reached a mean volume of -177 mm3, mice were randomized into treatment groups and injected intravenously with a single dose of test article at day 0 (see Table 20.1). Tumor volumes and body weights were monitored twice weekly over a 28-day study period. Supplemental Gel-Diet was given to all mice from day 4 to the end of study. Whole blood was collected retro-orbitally at multiple timepoints and processed to serum for pharmacokinetic analysis.Table 20.1: Treatment GroupsNumber of Group Test Article Dose (mg / kg) Animals (n) 1 Vehicle, 10 mM Na acetate, 9% sucrose, pH 4.5 0 6 2 V38332-DL-1 DAR 8 6 6 3 V39794-DL-1 DAR 8 6 6 4 Palivizumab-DL- 1 DAR 8 6 6 5 Cofetuzumab pelidotin 2.5 6Results

[0504] The results are shown in Fig. 18. Plots for group means were terminated when > 20% mice were lost (e.g. humane endpoints reached due to body weight loss or tumors exceeding 2000 mm3). At 6 mg / kg, v39794-DL-l DAR 8 robustly inhibited tumor growth compared to vehicle control and demonstrated greater tumor growth inhibition compared to the anli-PTK7 monospecific ADC v38332-DL-l DAR 8 and palivizumab-DL- 1 DAR 8 administered at the same antibody-matched dose and compared to cofetuzumab pelidotin dosed at 2.5 mg / kg.EXAMPLE 21: IN VIVO EFFICACY OF ANTI-PTK7 BIPARATOPIC ANTIBODYDRUG CONJUGATE IN PATIENT-DERIVED XENOGRAFT (PDX) MODELS

[0505] The in vivo efficacy of ADCs comprising the biparatopic anti-PTK7 antibody, v39794, conjugated to DL-1 at DAR 4 or DAR 8 (see Example 15) was assessed in PDX models of PTK7-expressing triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) as described below. PTK7 expression by each PDX model was confirmed by a research-grade immunohistochemistry (IHC) protocol. The anti-tumour activity of the anti-PTK7 biparatopic ADCs was compared to an ADC comprising the anti-PTK7 monospecific antibody, v41357, conjugated to DL-1 at DAR 8 (prepared following the protocol described in Example 15). An ADC comprising a non -targeting isotype control (palivizumab) antibody conjugated to DL-1 at DAR 8 and cofetuzumab pelidotin were included as negative and positive controls, respectively. The dose selected for administration of the anti-PTK7 biparatopic and monospecific ADCs was based on clinically relevant doses for known ADCs comprising topoisomerase 1 inhibitor payloads. The dose selected for administration of cofetuzumab pelidotin was based on the published clinically relevant dose.

[0506] Tumor fragments (approximately 2 to 3 mm diameter) from stock mice bearing patient-derived xenografts (HuPrime® Xenograft Models, Crown Bioscience Inc.) were implanted subcutaneously into the right flank of 6 - 8-week-old female BALB / c nude mice (see Table 21.1). When tumors reached a mean volume of approximately 155 - 176 mm3, mice were randomized into treatment groups and injected intravenously with a single dose of test article at day 0 as described in Table 21.2. Tumor volumes and body weights were monitored twice weekly over a 28-day study period as described in Example 20. Whole blood was collected from orbital sinus at day 0, 7 and 14 and processed to serum for future bioanalysis. Dietary gel supplements were provided to all study mice in the LU6408 model from Day 17 to Day 27.Table 2...

Claims

1. WE CLAIM:

1. An antibody construct comprising:a first antigen-binding domain that binds to human PTK7 and comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36, anda second antigen-binding domain that binds to human PTK7 and comprises the heavy chain CDR sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the light chain CDR sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64, wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

2. The antibody construct according to claim 1, wherein:(a) the first antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27; and / or(b) the second antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in anyone of SEQ ID NOs: 51, 54 or 56, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 52 or 57.

3. The antibody construct according to claim 1 or claim 2, wherein:the first antigen-binding domain comprises a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 2, and / orthe first antigen-binding domain comprises a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 3, and / orthe second antigen-binding domain comprises a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 4, and / orthe second antigen-binding domain comprises a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 5.

4. The antibody construct according to any one of claims 1 to 3, wherein:the first antigen-binding domain comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33, and / orthe first antigen-binding domain comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36, and / orthe second antigen-binding domain comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61, and / orthe second antigen-binding domain comprises a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64.

5. The antibody construct according to claim 1 or claim 2, wherein:the first antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, and / or the first antigen-binding domain comprises a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 34, and / or the second antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58, and / or the second antigen-binding domain comprises a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO: 62.

6. The antibody construct according to any one of claims 1 to 5, wherein the first and second antigen-binding domains are each a Fab comprising a CHI domain and a CL domain.

7. The antibody construct according to claim 6, wherein the CHI and CL domains of each Fab comprise sets of amino acid mutations to drive correct pairing between the CH1 domain and CL domain of the first antigen-binding domain and between the CH1 domain and the CL domain of the second antigen-binding domain.

8. The antibody construct according to any one of claims 1 to 7 further comprising a scaffold, wherein the first and second antigen-binding domains are operably linked to the scaffold, wherein the scaffold comprises an IgG Fc region.

9. The antibody construct according to claim 8, wherein the IgG Fc region is an IgGl Fc region.

10. The antibody construct according to claim 8 or claim 9, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein the Fc polypeptides comprise one or more amino acid substitutions to promote formation of the heterodimeric Fc region.

11. An antibody construct comprising:a first antigen-binding domain that binds to human PTK7 and comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 73, and a light chain comprising the sequence as set forth in SEQ ID NO: 75, anda second antigen-binding domain that binds to human PTK7 and comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 74, and a light chain comprising the sequence as set forth in SEQ ID NO: 76.

12. An antibody construct comprising an antigen-binding domain that binds to human PTK7, wherein the antigen-binding domain comprises the CDR (HCDR1, HCDR2, HCDR3) sequences of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 2, 31, 32 or 33, and the CDR (LCDR1, LCDR2, LCDR3) sequences of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 3, 34, 35 or 36, and wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

13. The antibody construct according to claim 12, wherein the antigen-binding domain comprises an HCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 7, 10, 13, 15 or 17; an HCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 8, 11, 14, 16 or 18; an HCDR3 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 9, 12 or 19; a LCDR1 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 20, 23 or 25; a LCDR2 comprising a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 21, 24 or 26, and a LCDR3 comprising a sequence selected from the amino acid sequences as set forth in SEQ ID NO: 22 or 27.

14. The antibody construct according to claim 12 or claim 13, wherein the antigen-binding domain comprises:(a) a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 2, and / or a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 3, or(b) a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast98%, atleast99%, or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 31, 32 or 33, and / or a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 34, 35 or 36.

15. The antibody construct according to claim 12 or claim 13, wherein the antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 32, and / or a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO:

34.

16. An antibody construct comprising an antigen-binding domain that binds to human PTK7, wherein the antigen-binding domain comprises the CDR (HCDR1, HCDR2, HCDR3) sequences of the VH domain having a sequence as set forth in any one of SEQ ID NOs: 4, 58, 59, 60 or 61, and the CDR (LCDR1, LCDR2, LCDR3) sequences of the VL domain having a sequence as set forth in any one of SEQ ID NOs: 5, 62, 63 or 64, and wherein the CDR sequences are defined by any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems.

17. The antibody construct according to claim 16, wherein the antigen-binding domain comprises an HCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 37, 40, 43, 45 or 47; an HCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 38, 44, 46, 92 or 93; an HCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 42, 90 or 91; a LCDR1 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 50, 53 or 55; a LCDR2 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 51, 54 or 56, and a LCDR3 having a sequence selected from the amino acid sequences as set forth in any one of SEQ ID NOs: 52 or 57.

18. The antibody construct according to claim 16 or claim 17, wherein the antigen-binding domain comprises:(a) a VH sequence that is a humanized version of the VH sequence as set forth in SEQ ID NO: 4, and / or a VL sequence that is a humanized version of the VL sequence as set forth in SEQ ID NO: 5, or(b) a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast98%, atleast99%,or 100% identical to the VH sequence as set forth in any one of SEQ ID NOs: 58, 59, 60 or 61, and / or a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in any one of SEQ ID NOs: 62, 63 or 64.

19. The antibody construct according to claim 16 or claim 17, wherein the antigen-binding domain comprises a VH sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence as set forth in SEQ ID NO: 58, and / or a VL sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence as set forth in SEQ ID NO:

62.

20. The antibody construct according to any one of claims 12 to 19, wherein the antigen-binding domain is a Fab or an scFv.

21. The antibody construct according to any one of claims 12 to 20, further comprising a scaffold, wherein the antigen-binding domain is operably linked to the scaffold, wherein the scaffold comprises an IgG Fc region.

22. The antibody construct according to any one of claims 12 to 21, further comprising a second antigen-binding domain.

23. The antibody construct according to claim 22, wherein the second antigen-binding domain binds to PTK7.

24. A polynucleotide or set of polynucleotides encoding the antibody construct according to any one of claims 1 to 23.

25. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to claim 24.

26. A host cell comprising the polynucleotide or set of polynucleotides according to claim 24 or the expression vector or set of expression vectors according to claim 25.

27. An antibody-drug conjugate comprising the antibody construct according to any one of claims 1 to 23 conjugated to one or more drug moieties.

28. An antibody-drug conjugate having general Formula (I):A-(L-(D)m)n (I)wherein:A is the antibody construct according to any one of claims 1 to 23;L is a linker;D is a drug moiety;m is between 1 and about 8, andn is 1 and about 12.

29. The antibody-drug conjugate according to claim 28, wherein:(a) m is 1 or 2, and / or(b) n is between about 2 and about 8, or n is between about 4 and about 8.

30. The antibody-drug conjugate according to any one of claims 27 to 29, wherein the drug moiety is a maytansinoid, maytansinoid analogue, benzodiazepine, pyrrolobenzodiazepine, duocarmycin, calicheamicin, calicheamicin analogue, auristatin, auristatin analogue, hemiasterlin, hemiasterlin analogue, tubulysin, tubulysin analogue, amatoxin, amatoxin analogue, camptothecin, camptothecin analogue, eribulin, TLR agonist or STING agonist.

31. The antibody-drug conjugate according to claim 28 or claim 29, wherein the drug moiety is:wherein * is the point of attachment to linker, L.

32. The antibody-drug conjugate according to any one of claims 28, 29 and 31, wherein linker, L, has:(a) Formula (II):'-z s,4qAAiAA2xt%(II)wherein:Z is a linking group that joins the linker to a target group on the antibody construct, A; Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site;X is a self-immolative group;q is 0 or 1;r is 1, 2 or 3;s is 0, 1 or 2;# is the point of attachment to the antibody construct, A, and% is the point of attachment to the drug moiety, D,or (b) Formula (V):'-Z S,,AA’AA^Y]7%(V)wherein:Z is a linking group that joins the linker to a target group on the anti-Ly6E antibody construct, A;Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]r forms a protease cleavage site;Y is -NH-CH2- or -NH-CH2-C(O)-;q is 0 or 1;r is 1, 2 or 3;v is 0 or 1;# is the point of attachment to the anti-Ly6E antibody construct, A, and% is the point of attachment to the drug moiety, D.

33. The antibody-drug conjugate according to claim 28 or claim 29, wherein L-(D)mhas the structure:where ** is the point of conjugation to the antibody construct, A.

34. A method of preparing the antibody-drug conjugate according to any one of 28, 29 and 31 comprising conjugating drug-linker L-(D)mto the antibody construct, A.

35. An antibody-drug conjugate having the structure:wherein n is between about 4 and about 8, andwherein A is an antibody construct comprising:(a) the first antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 10; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 11; anHCDR3 comprising a sequence as set forth in SEQ ID NO: 12; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 23; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 24, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 22; and(b) the second antigen-binding domain comprises an HCDR1 comprising a sequence as set forth in SEQ ID NO: 40; an HCDR2 comprising a sequence as set forth in SEQ ID NO: 92; an HCDR3 comprising a sequence as set forth in SEQ ID NO: 42; a LCDR1 comprising a sequence as set forth in SEQ ID NO: 53; a LCDR2 comprising a sequence as set forth in SEQ ID NO: 54, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 52.

36. The antibody-drug conjugate according to claim 35, wherein:(a) the first antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 32, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 34, and(b) the second antigen-binding domain comprises a VH sequence comprising the sequence as set forth in SEQ ID NO: 58, and a VL sequence comprising the sequence as set forth in SEQ ID NO: 62.

37. The antibody-drug conjugate according to claim 35 or claim 36, wherein the first and second antigen-binding domains are each a Fab comprising a CHI domain and a CL domain.

38. The antibody-drug conjugate according to claim 37, wherein the CHI and CL domains of each Fab comprise sets of amino acid mutations to drive correct pairing between the CH1 domain and CL domain of the first antigen-binding domain and between the CH1 domain and the CL domain of the second antigen-binding domain.

39. The antibody-drug conjugate according to claim 38, wherein:(a) the CHI domain and CL domain of one of the first or second antigen-binding domains comprise the set of amino acid mutations:CH1: A139W_L143E_K145T_Q179ECL: F116A_Q124R_L135V_T178Rand the CHI domain and CL domain of the other antigen-binding domain comprise the set of amino acid mutations:CH1: Q179RCL: Q124E_L135W_T178E_T180E, or(b) the CHI domain and CL domain of one of the first or second antigen-binding domains comprise the set of amino acid mutations:CH1: L143RCL: Q124E_V133Eand the CHI domain and CL domain of the other antigen-binding domain comprise the set of amino acid mutations:CH1: L143E_K145T_Q179ECL: Q124R_T178R.

40. The antibody-drug conjugate according to any one of claims 35 to 39 further comprising a scaffold, wherein the first and second antigen-binding domains are operably linked to the scaffold, and wherein the scaffold comprises an IgG Fc region.

41. The antibody-drug conjugate according to claim 40, wherein the IgG Fc region is an IgGl Fc region.

42. The antibody construct according to claim 40 or claim 41, wherein the IgG Fc region is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein the Fc polypeptides comprise one or more amino acid substitutions to promote formation of the heterodimeric Fc region.

43. The antibody-drug conjugate according to claim 42, wherein the amino acid substitutions comprised by the Fc polypeptides are:(a) the amino acid substitutions L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T366L_K392M_T394W in the other Fc polypeptide, or(b) the amino acid substitutions L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T366L_K392L_T394W in the other Fc polypeptide, or(c) the amino acid substitutions T350V_L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392L_T394W in the other Fc polypeptide, or (d) the amino acid substitutions T350V_L351Y_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392M_T394W in the other Fc polypeptide, or(e) the amino acid substitutions T350V_L351Y_S400E_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_N390R_K392M_T394W in the other Fc polypeptide,wherein the numbering of amino acids is according to the EU index44. The antibody-drug conjugate according to claim 35, wherein the first antigen-binding domain comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 73, and a light chain comprising the sequence as set forth in SEQ ID NO: 75, and the second antigen-binding domain comprises a heavy chain comprising the sequence as set forth in SEQ ID NO: 74, and a light chain comprising the sequence as set forth in SEQ ID NO: 76.

45. A method of preparing the antibody-drug conjugate according to any one of 35 to 44 comprising conjugating a drug-linker having the structure:to the antibody construct, A, wherein the drug-linker is conjugated to native cysteine residues of the antibody construct, A.

46. A pharmaceutical composition comprising the antibody construct according to any one of claims 1 to 23, or the antibody-drug conjugate according to any one of claims 27 to 33 and 35 to 44, and a pharmaceutically acceptable carrier or diluent.

47. An antibody construct according to any one of claims 1 to 23, or the antibody-drug conjugate according to any one of claims 27 to 33 and 35 to 44, for use in therapy.

48. The antibody construct for use or the antibody-drug conjugate for use according to claim 47, wherein the therapy comprises treatment of cancer.

49. Use of an antibody construct according to any one of claims 1 to 23, or the antibody-drug conjugate according to any one of claims 27 to 33 and 35 to 44, in the manufacture of a medicament for the treatment of cancer.

50. A method of inhibiting the growth of tumor cells comprising contacting the cells with an antibody construct according to any one of claims 1 to 23, or the antibody-drug conjugate according to any one of claims 27 to 33 and 35 to 44.

51. A method of treating a subject having a cancer comprising administering to the subject an effective amount of the antibody construct according to any one of claims 1 to 23, or the antibodydrug conjugate according to any one of claims 27 to 33 and 35 to 44.

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