PTK7-binding antibodies

Abstract

The present disclosure provides humanised internalising antibodies that bind to PTK7, and methods of using them to treat cancer. The epitopes groups of the anti-PTK7 antibodies are identified.

Description

Docket No. 122878.WO016PTK7-BINDING PROTEINSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application 63 / 722,437, filed November 19, 2024, and U.S. Patent Application No. 19 / 175,619, filed April 10, 2025, the disclosures of which are incorporated by reference herein in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is part of the specification, and is hereby incorporated by reference herein in its entirety. The electronic copy of the Sequence Listing, created on November 11, 2025, is named 122878. WO016.xml and is 320,717 bytes in size.BACKGROUND OF THE INVENTION

[0003] Protein tyrosine kinase 7 (PTK7), also known as colon carcinoma kinase 4 (CCK4), is a receptor tyrosine kinase involved in non-canonical Wnt signaling and was first identified as a gene upregulated in colon cancer cells. PTK7 belongs to the Wnt ligand binding receptor family whose other members include receptor tyrosine kinase-like orphan receptors 1 (ROR1) and 2 (ROR2), and receptor tyrosine kinase RYK. Members of this receptor family are transmembrane proteins that are void of kinase activity. PTK7 plays roles in embryonic development, maintenance of tissue homeostasis, and stem cell signaling; however, expression of PTK7 is absent or minimal in differentiated healthy adult tissues. Overexpression of PTK7 has been reported in a variety of cancers including head and neck cancer, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), bladder cancer, and ovarian cancer. In this milieu, PTK7 promotes cell survival, migration, and invasion, and chemotherapy resistance.

[0004] PTK7 expression has been associated with poor prognosis and higher metastatic potential in patients with various cancers, and thus represents a promising therapeutic target. In view of PTK7’s critical role in tumor progression, there is a need for new and improved cancer immunotherapies that target PTK7.SUMMARY OF THE INVENTION

[0005] The present disclosure provides recombinant proteins that bind specifically to PTK7. In some embodiments, the present PTK7-binding protein is an anti-PTK7 antibody or an antigen-binding portion thereof.

[0006] In some embodiments, the anti-PTK7 antibody or antigen-binding portion thereof comprises heavy chain CDR (HCDR) 1-3 and light chain CDR (LCDR) 1-3 comprising SEQ ID NOs: 151, 157, 161, 164, 167, and 170, respectively. In some embodiments, the anti- PTK7 antibody or antigen-binding portion thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising SEQ ID NOs:76 and 75, respectively.

[0007] In some embodiments, the anti-PTK7 antibody or antigen-binding portion thereof comprises a VH that comprises SEQ ID NOs: 154, 155, and 161, and a VL that comprises SEQ ID NOs: 163, 166, and 170. In some embodiments, the anti-PTK7 antibody or antigenbinding portion is humanized. In certain embodiments, the anti-PTK7 antibody or antigenbinding portion thereof comprises a VH that comprises framework regions derived from a human IGHV1 -46*01 germline gene. The VH may, for example, comprise one or more back mutations selected from Y91F, R71 V, V78A, M48I, M69L, and V20L, wherein the numbering is according to SEQ ID NO: 172. In certain embodiments, additionally or alternatively, the anti-PTK7 antibody or antigen-binding portion thereof comprises a VL that comprises framework regions derived from a human IGKV1D- 16*01 germline gene. The VL may, for example, comprise one or more back mutations selected from I21L, Y36L, A43T, S46R, G66R, T69S, and F71Y, wherein the numbering is according to SEQ ID NO: 183.

[0008] In some embodiments, the anti-PTK7 antibody or antigen-binding portion thereof comprises a) a VH that comprises any one of SEQ ID NOs: 172-182, 193, 196, and 197, or an amino acid sequence that is at least 95% identical thereto; b) a VL that comprises any one of SEQ ID NOs: 183-192, 194, and 195, or an amino acid sequence that is at least 95% identical thereto; or c) a) and b).In particular embodiments, the anti-PTK7 antibody or antigen-binding portion thereof comprises a VH and VL that comprise SEQ ID NOs: 178 and 188, respectively, SEQ ID NOs: 193 and 188, respectively, or SEQ ID NOs: 173 and 188, respectively.

[0009] In some embodiments, the anti-PTK7 antibody or antigen-binding portion thereof, the antibody comprises a human IgGi constant region. The human IgGi constant region may comprise, for example, SEQ ID NO:201, optionally without the C-terminal lysine. In someembodiments, additionally or alternatively, the anti-PTK7 antibody or antigen-binding portion thereof comprises a human light chain constant region that comprises SEQ ID NO: 199.

[0010] The present disclosure also provides an isolated anti-PTK7 antibody comprising a heavy chain that comprises the amino acid sequences of SEQ ID NOs: 193 and 201, optionally without the C-terminal lysine, and a light chain that comprises the amino acid sequences or SEQ ID NOs: 188 and 199.

[0011] The present disclosure also provides an isolated anti-PTK7 antibody that comprises a) HCDR1-3 and LCDR1-3 comprising SEQ ID NOs:204, 209, 213, 216, 219, and 222, respectively; b) VH and VL comprising SEQ ID NOs: 112 and 111, respectively; or c) an HC comprising SEQ ID NOs: 112 and 201, optionally without the C- terminal lysine, and an LC comprising SEQ ID NOs: 111 and 199.

[0012] The present disclosure also provides an isolated anti-PTK7 antibody that comprises a) HCDR1-3 and LCDR1-3 comprising SEQ ID NOs:226, 231, 235, 238, 241, and 244, respectively; b) VH and VL comprising SEQ ID NOs: 128 and 127, respectively; or c) an HC comprising SEQ ID NOs: 128 and 201, optionally without the C- terminal lysine, and an LC comprising SEQ ID NOs: 127 and 199.

[0013] In some embodiments, the isolated anti-PTK7 antibody or antigen-binding portion thereof is conjugated to a cytotoxin or a detectable label.

[0014] The present disclosure also provides pharmaceutical compositions comprising an anti-PTK7 antibody or antigen-binding portion thereof as described herein and a pharmaceutically acceptable excipient.

[0015] The present disclosure also provides nucleic acid molecule(s) encoding an anti- PTK7 antibody or antigen-binding portion thereof as described herein, as well as expression vector(s) comprising the nucleic acid molecule(s) and recombinant host cells comprising the expression vector(s). Also provided is a method of making an anti-PTK7 antibody or an antigen-binding portion thereof, comprising: culturing a recombinant host cell described herein under conditions that allow expression of the antibody or portion, and isolating the antibody or portion from the cell culture.

[0016] The present disclosure also provides a method of treating a PTK7-positive cancer in a patient in need thereof (e.g., a human patient), comprising administering to the patient an anti-PTK7 antibody or antigen-binding portion thereof described herein or a pharmaceutical composition described herein. Also provided are use of an anti-PTK7 antibody or antigenbinding portion thereof described herein for the manufacture of a medicament for treating a PTK7-positive cancer in a patient in need thereof, or an anti-PTK7 antibody or antigenbinding portion thereof described herein or a pharmaceutical composition described herein for use in treating a PTK7-positive cancer in a patient in need thereof. In certain embodiments, the PTK7-positive cancer is selected from head and neck cancer, non-small cell lung cancer, small-cell lung cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, and sarcoma.

[0017] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0018] FIG. 1 shows an overview of the epitope groups (epitope bins) identified by binding competition analysis of a panel of 42 anti-PTK7 antibodies. Antibodies connected by black lines indicate cross blocking activity in both orientations (ligand and analyte). Antibodies connected by dashed lines indicate unidirectional blocking (antibody is blocked when tested as an analyte only, for example). Antibodies are grouped according to competition patterns with other anti-PTK7 antibodies. The antibodies are separated into four major groupings and are further are categorized into eighteen communities (shaded outlines).

[0019] FIG. 2 is a line graph demonstrating the median mean fluorescence intensity (MFI) as a measure of binding of the listed antibodies to NCI-H520 cells.

[0020] FIG. 3 is a line graph showing binding (median MFI) of the listed antibodies to NCI-H520 cells.

[0021] FIG. 4 is a line graph showing binding (median MFI) of the listed antibodies to NCI-H520 cells.

[0022] FIG. 5 is a line graph showing the binding (median MFI) of the listed antibodies to Jurkat cells.

[0023] FIG. 6 is a line graph demonstrating the internalization of the listed antibodies in NCI-H520 cells as measured by the loss of surface binding signal over time.

[0024] FIG. 7 is a line graph demonstrating the internalization of the listed antibodies in NCI-H520 cells as measured by the loss of surface binding signal over time.

[0025] FIG. 8 is a line graph demonstrating the internalization of the listed antibodies in HCI-H520 cells as measured by the loss of surface binding signal over time.

[0026] FIG. 9A is a pair of tables showing different definitions of CDRs for antibody SLX-1040. SEQ: SEQ ID NO. Grafted Seq.: sequence grafted to human acceptor frameworks during humanization.

[0027] FIG. 9B is a sequence alignment of the heavy and light chain variable domains of antibody SLX-1040 (“18327”) (SEQ ID NOs: 76 and 75, respectively) with the most homologous human heavy and light chain germline sequences (IGHV1 -46*01 and IGKV1D- 16*01, respectively) (CDR graft sequences shown in SEQ ID NOs: 172 and 183, respectively). CDRs to be grafted to human acceptor sequences for humanization are in boldface. Asterisks indicate potential positions for making back mutations during humanization.

[0028] FIGs. 9C and 9D are tables showing humanized versions of SLX-1040 with various combinations of VH and VL variants having the indicated framework back mutations. SEQ: SEQ ID NO.

[0029] FIG. 10 is a line graph showing binding (MFI) of the listed antibodies to NCI- 11520 cells.

[0030] FIG. 11 is a line graph showing binding (MFI) of the listed antibodies to CHO cells expressing cynomolgus PTK7.

[0031] FIG. 12 is a line graph showing binding (MFI) of the listed antibodies to NCI- 11520 cells expressing cynomolgus PTK7.

[0032] FIG. 13 is a line graph showing binding (MFI) of the listed antibodies to CHO cells expressing cynomolgus PTK7.

[0033] FIG. 14 is a line graph showing binding (MFI) of the listed antibodies to NCI- H520 cells.

[0034] FIG. 15 is a line graph showing binding (MFI) of the listed antibodies to rhesus CMMT cells.

[0035] FIG. 16 is a line graph demonstrating the internalization of the listed antibodies in NCLH520 cells as measured by the loss of surface binding signal over time.

[0036] FIG. 17 is a table showing molecular stability data of antibody variants after freeze / thaw cycles.

[0037] FIG. 18 is a table showing molecular stability data of antibody variants after thermal stress.DETAILED DESCRIPTION OF THE INVENTION

[0038] The present disclosure provides novel proteins that specifically bind to PTK7 (“PTK7-binding proteins”). These proteins may be antibodies or comprise antigen-binding portions thereof. The proteins can be used to treat cancer (e.g., PTK7-positive cancer) in patients in need thereof.

[0039] The term “antibody” (Ab) or “immunoglobulin” (Ig), as used herein, refers to a tetramer comprising two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (VL) and a light chain constant region (CL). The VH and VL domains can be subdivided further into regions of hypervariability, termed “complementarity determining regions” (CDRs), interspersed with regions that are more conserved, termed “framework regions” (FRs). Each VH and VL is composed of three CDRs (HCDR herein designates a CDR from the heavy chain; and LCDR herein designates a CDR from the light chain) and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acid numbers, and of FR and CDR regions, in the heavy or light chain may be in accordance with IMGT® definitions (Lefranc et al., Dev Comp Immunol. (2003) 27(l):55-77); or the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J Mol Biol. (1987) 196:901-17; Chothia et al., Nature (1989) 342:878-83; Abhinandan et al., Molecular Immunology (2008) 45(14):3832-39; MacCallum et al., J Mol Biol. (1996) 262:732-45; or Honegger and Pliickthun, J Mol Biol. (2001) 309(3):657-70. The CDR boundaries of various schemes are illustrated in Table 1, where the amino acid numbers are Kabat numbers unless otherwise indicated.Table 1. CDR Delineations According to Various Schemes

[0040] The term “recombinant antibody” or “recombinant protein” refers to a non- naturally occurring antibody or protein that is expressed from a cell or cell line comprising nucleotide sequence(s) that encode the antibody or protein, wherein said nucleotide sequence(s) are not naturally associated with the cell.

[0041] The term “isolated protein,” “isolated polypeptide” or “isolated antibody” refers to a protein, polypeptide or antibody that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, and / or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

[0042] The term “affinity” refers to a measure of the attraction between an antigen and an antibody. The intrinsic attractiveness of the antibody for the antigen is typically expressed as the binding affinity equilibrium constant (KD) of a particular antibody-antigen interaction.An antibody is said to specifically bind to an antigen when the KD for the binding is — 1 pM, e.g., — 100 nM or — 10 nM. A KD binding affinity constant can be measured, e.g., by surface plasmon resonance (Biacore™) using the Biacore™ system, the IBIS MX96 SPR system from IBIS Technologies or the Carterra LSA SPR platform, or by Bio-Layer Interferometry, for example using the Octet™ system from ForteBio.

[0043] The term “epitope” as used herein refers to a portion (determinant) of an antigen that specifically binds to an antibody or a related molecule such as a bi-specific binding molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be “linear” or “conformational.” In a linear epitope, all of the points of interaction between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformationalepitope, the points of interaction occur across amino acid residues on the protein that are separated from one another in the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope using techniques well known in the art. For example, an antibody to a linear epitope may be generated, e.g., by immunizing an animal with a peptide having the amino acid residues of the linear epitope. An antibody to a conformational epitope may be generated, e.g., by immunizing an animal with a mini-domain containing the relevant amino acid residues of the conformational epitope. An antibody to a particular epitope can also be generated, e.g., by immunizing an animal with the target molecule of interest (e.g., PTK7) or a relevant portion thereof, then screening for binding to the epitope. An antibody to a particular epitope also may be generated using phage display methods.

[0044] One can determine whether an antibody binds to the same epitope as or competes for binding with an anti-PTK7 antibody of the invention by using methods known in the art, including, without limitation, competition assays, epitope binning, and alanine scanning. In one embodiment, one allows the anti-PTK7 antibody of the invention to bind to PTK7 under saturating conditions, and then measures the ability of the test antibody to bind to PTK7. If the test antibody is able to bind to PTK7 at the same time as the reference anti-PTK7 antibody, then the test antibody binds to a different epitope than the reference anti-PTK7 antibody. However, if the test antibody is not able to bind to PTK7 at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the anti-PTK7 antibody of the invention. This experiment can be performed using, e.g., ELISA, RIA, Biacore™, SPR, Bio-Layer Interferometry or flow cytometry. To test whether an anti-PTK7 antibody cross-competes with another anti- PTK7 antibody, one may use the competition method described above in two directions, i.e., determining if the known antibody blocks the test antibody and vice versa. Such crosscompetition experiments may be performed, e.g., using an IBIS MX96 or Carterra LSA SPR instrument or the Octet™ system.

[0045] The term “antigen-binding portion” of an antibody (or simply “antibody portion”), as used herein, refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human PTK7, or a portion thereof). It has been shown that certain fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” include (i) a Fab fragment: a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment: a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) capable of specifically binding to an antigen. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (scFv)). Also within the present disclosure are antigen-binding molecules comprising a VH and / or a VL. In the case of a VH, the molecule may also comprise one or more of a CHI, hinge, CH2, or CH3 region. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bi-specific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigenbinding sites.

[0046] Antibody portions, such as Fab and F(ab’)2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, e.g., as described herein.

[0047] In some embodiments, antibodies may originate from non-human sources and have been humanized to reduce anti-antibody immune responses. The term “humanize” refers to the fact that where an antibody is wholly or partially of non-human origin (for example, a murine or chicken antibody obtained from immunization of mice or chickens, respectively, with an antigen of interest, or a chimeric antibody based on such a murine or chicken antibody), it is possible to replace certain amino acids, in particular in the framework regions and constant regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. Although it is not possible to precisely predict the immunogenicity, and thereby the human anti-antibody response, of a particular antibody, non-human antibodies tend to be more immunogenic in humans than human antibodies. Antibodies of non-human origin thus can be humanized to reduce the risk of a human antiantibody response.

[0048] Humanization typically involves modification of the framework regions of the variable domain sequences. Amino acid residues that are part of complementarity determining regions (CDRs) most often will not be altered in connection with humanization, although in certain cases it may be desirable to alter individual CDR amino acid residues, for example to remove a glycosylation site, a deamidation site, an aspartate isomerization site or an undesired cysteine or methionine residue. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of an N-glycosylation site may be achieved by mutating either the Asn or the Ser / Thr residue to a different residue, preferably by way of conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent in other dipeptide sequences such as Asn-Ser, Asn-Thr, and Asn-Ala. When such a deamidation site, in particular Asn-Gly, is present in a CDR sequence, it may therefore be desirable to remove the site, typically by conservative substitution to remove one of the implicated residues.

[0049] The class (isotype) and subclass of anti-PTK7 antibodies may be determined by any method known in the art. In general, the class and subclass of an antibody may be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are available commercially. The class and subclass can be determined by ELISA or Western blot as well as other techniques. Alternatively, the class and subclass may be determined by sequencing all or a portion of the constant regions of the heavy and / or light chains of the antibodies, comparing their amino acid sequences to the known amino acid sequences of various classes and subclasses of immunoglobulins, and determining the class and subclass of the antibodies.

[0050] The term “monoclonal antibody” or “monoclonal antibody composition” means a preparation of antibody molecules of single molecular composition, which displays a single binding specificity and affinity for a particular epitope.

[0051] Unless otherwise indicated, all antibody variable region amino acid residue numbers referred to in this disclosure are those under the Kabat numbering scheme. Eu numbering is employed for constant regions.I. PTK7-Binding Proteins

[0052] The present disclosure provides PTK-binding proteins (e.g., anti-PTK7 antibodies and proteins comprising the antibodies or antigen-binding portions thereof). Unless otherwise stated, “PTK7” refers to human PTK7 herein. A human PTK7 polypeptide sequence is available under UniProt Accession No. Q13308 (PTK7 HUMAN) and is shown below:MGAARGSPAR PRRLPLLSVL LLPLLGGTQT AIVFIKQPSS QDALQGRRAL LRCEVEAPGP VHVYWLLDGA PVQDTERRFA QGSSLSFAAV DRLQDSGTFQ CVARDDVTGE E ARSANAS FN IKWIEAGPVV LKHPASEAEI QPQTQVTLRC HIDGHPRPTY QWFRDGTPLS DGQSNHTVSS KERNLTLRPA GPEHSGLYSC CAHSAFGQAC SSQNFTLSIA DESFARVVLA PQDVVVARYE EAMFHCQFSA QPPPSLQWLF EDETPITNRS RPPHLRRATV FANGSLLLTQ VRPRNAGIYR CIGQGQRGPP I ILEATLHLA EIEDMPLFEP RVFTAGSEER VTCLPPKGLP EPSVWWEHAG VRLPTHGRVY QKGHELVLAN IAESDAGVYT CHAANLAGQR RQDVNITVAT VPSWLKKPQD SQLEEGKPGY LDCLTQATPK PTVVWYRNQM LISEDSRFEV FKNGTLRINS VEVYDGTWYR CMSSTPAGSI EAQARVQVLE KLKFTPPPQP QQCMEFDKEA TVPCSATGRE KPTIKWERAD GSSLPEWVTD NAGTLHFARV TRDDAGNYTC IASNGPQGQI RAHVQLTVAV FITFKVEPER TTVYQGHTAL LQCEAQGDPK PLIQWKGKDR ILDPTKLGPR MHI FQNGSLV IHDVAPEDSG RYTCIAGNSC NIKHTEAPLY VVDKPVPEES EGPGSPPPYK MIQTIGLSVG AAVAYIIAVL GLMFYCKKRC KAKRLQKQPE GEEPEMECLN GGPLQNGQPS AEIQEEVALT SLGSGPAATN KRHSTSDKMH FPRSSLQPIT TLGKSEFGEV FLAKAQGLEE GVAETLVLVK SLQSKDEQQQ LDFRRELEMF GKLNHANVVR LLGLCREAEP HYMVLEYVDL GDLKQFLRIS KSKDEKLKSQ PLSTKQKVAL CTQVALGMEH LSNNRFVHKD LAARNCLVSA QRQVKVSALG LSKDVYNSEY YHFRQA VPL RWMSPEAILE GDFSTKSDVW AFGVLMWEVF THGEMPHGGQ ADDEVLADLQ AGKARLPQPE GCPSKLYRLM QRCWALSPKD RPSFSEIASA LGDSTVDSKP ( SEQ ID NO 198 )In the above sequence, amino acids 1-30 correspond to the signal sequence; amino acids 31- 704 correspond to the extracellular domain (ECD); amino acids 705-725 correspond to the transmembrane domain; and amino acids 726-1070 correspond to the intracellular domain. The PTK7 binders herein bind to the ECD of PTK7.

[0053] The present disclosure provides antibodies directed against PTK7, and antigenbinding portions thereof. One advantage of the novel anti-PTK7 antibodies of the invention is that they are able to effectively bind to tumor cells as measured by increased mean fluorescence intensity (MFI) in cells exposed to the different antibody candidates; see, e.g., Examples 4 and 11. Furthermore, the novel anti-PTK7 antibodies of the invention are able to be effectively internalized in tumor cell lines as measured by the decrease in surface bound antibody over time, as demonstrated in Examples 5 and 12.

[0054] In some embodiments, the present disclosure provides an anti-PTK7 antibody or an antigen-binding portion thereof that competes or cross-competes for binding to human PTK7 with, or binds to the same epitope of human PTK7 as, any one of SLX-1040, Z12, Z23, Z31,SLX-1042, or SLX-1059.

[0055] The present disclosure also provides an anti-PTK7 antibody or an antigen-binding portion thereof that competes or cross-competes for binding to human PTK7 with, or binds to the same epitope of human PTK7 as, an antibody comprising: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:76 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:75; b) a VH comprising the amino acid sequence of SEQ ID NO: 178 and a VL comprising the amino acid sequence of SEQ ID NO: 188; c) a VH comprising the amino acid sequence of SEQ ID NO: 193 and a VL comprising the amino acid sequence of SEQ ID NO: 188; d) a VH comprising the amino acid sequence of SEQ ID NO: 173 and a VL comprising the amino acid sequence of SEQ ID NO: 188; e) a VH comprising the amino acid sequence of SEQ ID NO: 112 and a VL comprising the amino acid sequence of SEQ ID NO: 111; or f) a VH comprising the amino acid sequence of SEQ ID NO: 128 and a VL comprising the amino acid sequence of SEQ ID NO: 127.

[0056] In some embodiments, the anti-PTK7 antibody has a heavy chain CDR1 (H- CDR1) that is or is at least 90% identical in sequence to any one of SEQ ID NO: 151 or 154, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 151 or 154.

[0057] In some embodiments, the anti-PTK7 antibody has a heavy chain CDR2 (H- CDR2) that is or is at least 90% identical in sequence to SEQ ID NO: 155 or 157, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 155 or 157.

[0058] In some embodiments, the anti-PTK7 antibody has a heavy chain CDR3 (H- CDR3) that is or is at least 90% identical in sequence to SEQ ID NO: 161, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 161.

[0059] In some embodiments, the anti-PTK7 antibody has a light chain CDR1 (L-CDR1) that is or is at least 90% identical in sequence to SEQ ID NO: 163 or 164, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 163 or 164.

[0060] In some embodiments, the anti-PTK7 antibody has a light chain CDR2 (L-CDR2) that is or is at least 90% identical in sequence to SEQ ID NO: 166 or 167, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 166 or 167.

[0061] In some embodiments, the anti-PTK7 antibody has a light chain CDR3 (L-CDR3) that is or is at least 90% identical in sequence to SEQ ID NO: 170, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 170.

[0062] Any combination of the above H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 sequences is also contemplated.

[0063] In some embodiments, the anti-PTK7 antibody or antigen-binding portion of the present disclosure comprises the HCDR1-3 and LCDR1-3 amino acid sequences of any one of the antibodies exemplified herein. The assignment of CDR regions may be in accordance with any method known in the art, such as IMGT®, Kabat, Chothia, Martin, Contact, or AHo definitions, or any combination of any of these definitions (Kabat plus Chothia, for example). Examples of CDR definitions under different methods are shown below for SLX-1040 (SEQ: SEQ ID NO):SLX-1040 HCDRsSLX-1040 LCDRsThus, for example, the SLX-1040 IMGT®-defined HCDR1-3 and LCDR1-3 sequences ofSEQ ID N0s:151, 157, 161, 164, 167, and 170, respectively, may be replaced in any embodiment described herein bySEQ ID NOs: 149, 155, 160, 163, 166, and 170, respectively;SEQ ID NOs: 150, 156, 160, 163, 166, and 170, respectively;SEQ ID NOs: 152, 158, 161, 163, 168, and 170, respectively; orSEQ ID NOs: 153, 159, 162, 165, 169, and 171, respectively.Also contemplated is a set of SLX-1040 CDRs wherein each of HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3 may individually be specified according to any of the methods for defining SLX-1040 CDRs as shown above (e.g., HCDR1 specified by the Kabat definition, HCDR2 specified by the Chothia definition, etc.). The same means for defining SLX-1040 CDRs are contemplated for any of the exemplified antibodies herein.

[0064] In some embodiments, the anti-PTK7 antibody or antigen-binding portion thereof has:- an HCDR1 selected from SEQ ID NOs: 149-154;- an HCDR2 selected from SEQ ID NOs: 155-159;- an HCDR3 selected from SEQ ID NOs: 160-162;- an LCDR1 selected from SEQ ID NOs: 163-165;- an LCDR2 selected from SEQ ID NOs: 166-169; and- an LCDR3 selected from SEQ ID NOs: 170 and 171.

[0065] In some embodiments, the anti-PTK7 antibody or antigen-binding portion thereof has HCDRl-3 and LCDRl-3 comprising SEQ ID NOs: 151, 157, 161, 164, 167, and 170, respectively. In some embodiments, the anti-PTK7 antibody or antigen-binding portion has HCDR1-3 and LCDRl-3 comprising SEQ ID NOs: 154, 155, 161, 163, 166, and 170, respectively.

[0066] In some embodiments, the anti-PTK7 antibody or antigen-binding portion has HCDR1-3 and LCDR1-3 comprising SEQ ID NOs:204, 209, 213, 216, 219, and 222, respectively.

[0067] In some embodiments, the anti-PTK7 antibody or antigen-binding portion has HCDR1-3 and LCDR1-3 comprising SEQ ID NOs:226, 231, 235, 238, 241, and 244, respectively.

[0068] In some embodiments, the anti-PTK7 antibody or antigen-binding portion thereof has a VH that is at least 90% identical in sequence to any one of SEQ ID NOs:76, 173, 178, and 193, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs:76, 173, 178, and 193. Additionally or alternatively, the anti- PTK7 antibody or antigen-binding portion thereof may have a VL that is at least 90% identical in sequence to SEQ ID NO:75 or 188, e.g. at least 92% identical, such as at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:75 or 188. Any combination of these VH and VL amino acid sequences is contemplated.

[0069] In some embodiments, the VH may be any one of SEQ ID NOs:76, 173, 178, and 193 and the VL may be SEQ ID NO:75 or 188. In some embodiments, the VH may be any one of SEQ ID NOs: 173, 178, and 193 and the VLmay be SEQ ID NO: 188.

[0070] In some embodiments, the antibody or portion has a VH comprising SEQ ID NO:76 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising SEQ ID NO:75 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the antibody or portion has a VH and a VL comprising SEQ ID NOs:76 and 75, respectively.

[0071] In some embodiments, the antibody or portion has a VH comprising SEQ ID NO: 178 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising SEQ ID NO: 188 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the antibody or portion has a VH and a VL comprising SEQ ID NOs: 178 and 188, respectively.

[0072] In some embodiments, the antibody or portion has a VH comprising SEQ ID NO: 193 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising SEQ ID NO: 188 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the antibody or portion has a VH and a VL comprising SEQ ID NOs: 193 and 188, respectively.

[0073] In some embodiments, the antibody or portion has a VH comprising SEQ ID NO: 173 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising SEQ ID NO: 188 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the antibody or portion has a VH and a VL comprising SEQ ID NOs: 173 and 188, respectively.

[0074] In some embodiments, the antibody or portion has a VH comprising SEQ ID NO: 112 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising SEQ ID NO: 111 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In certain embodiments, the antibody or portion has a VH and a VL comprising SEQ ID NOs: 112 and 111, respectively.

[0075] In some embodiments, the antibody or portion has a VH comprising SEQ ID NO: 128 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a VL comprising SEQ ID NO: 127 or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical thereto. In certain embodiments, the antibody or portion has a VH and a VL comprising SEQ ID NOs: 128 and 127 respectively.

[0076] In some embodiments, the anti-PTK7 antibody has a heavy chain (HC) amino acid sequence comprising a VH that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:76, 173, 178, or 193 and a heavy chain constant domain (CH) that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:201, optionally without the C-terminal lysine. Additionally or alternatively, the anti- PTK7 antibody may have a light chain (LC) amino acid sequence comprising a VL that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:75 or 188 and a light chain constant domain (CL) that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 199. Any combination of these HC and LC amino acid sequences is also contemplated.

[0077] In some embodiments, the anti-PTK7 antibody has an HC amino acid sequence comprising a VH that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 112 and a CH that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:201, optionally without the C-terminal lysine. Additionally or alternatively, the anti-PTK7 antibody may have an LC amino acid sequence comprising a VL that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 111 and a CL that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 199.

[0078] In some embodiments, the anti-PTK7 antibody has an HC amino acid sequence comprising a VH that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 128 and a heavy chain constant domain CH that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:201, optionally without the C-terminal lysine. Additionally or alternatively, the anti-PTK7 antibody may have an LC amino acid sequence comprising a VL that is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 127 and a CLthat is or is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 199.

[0079] In some embodiments, the anti-PTK7 antibody comprises an HC amino acid sequence comprising SEQ ID NOs:76 and 201, optionally without the C-terminal lysine, and an LC amino acid sequence comprising SEQ ID NOs:75 and 199.

[0080] In some embodiments, the anti-PTK7 antibody comprises an HC amino acid sequence comprising SEQ ID NOs: 178 and 201, optionally without the C-terminal lysine, and an LC amino acid sequence comprising SEQ ID NOs: 188 and 199.

[0081] In some embodiments, the anti-PTK7 antibody comprises an HC amino acid sequence comprising SEQ ID NOs: 193 and 201, optionally without the C-terminal lysine, and an LC amino acid sequence comprising SEQ ID NOs: 188 and 199.

[0082] In some embodiments, the anti-PTK7 antibody comprises an HC amino acid sequence comprising SEQ ID NOs: 173 and 201, optionally without the C-terminal lysine, and an LC amino acid sequence comprising SEQ ID NOs: 188 and 199.

[0083] In some embodiments, the anti-PTK7 antibody comprises an HC amino acid sequence comprising SEQ ID NOs: 112 and 201, optionally without the C-terminal lysine, and an LC amino acid sequence comprising SEQ ID NOs: 111 and 199.

[0084] In some embodiments, the anti-PTK7 antibody comprises an HC amino acid sequence comprising SEQ ID NOs: 128 and 201, optionally without the C-terminal lysine, and an LC amino acid sequence comprising SEQ ID NOs: 127 and 199.

[0085] The anti-PTK7 antibody of the present disclosure can be an IgG, an IgM, an IgE, an IgA, or an IgD molecule, but is typically of the IgG isotype, e.g., of IgG subclass IgGi, IgG2, IgGs or IgG4. In some embodiments, the antibody is of the isotype subclass IgGi.

[0086] In some embodiments, any of the anti-PTK7 antibodies may bind to human PTK7 with an ECso of no more than 200 nM, for example, no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 5, 1, 0.5, or 0.25 nM.

[0087] In some embodiments, any of the anti-PTK7 antibodies may be internalized in cells with a loss of cell surface binding signal over time of, for example, about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% loss of surface binding signal.

[0088] The present disclosure also contemplates an anti-PTK7 antibody or antigenbinding portion with any combination of the above properties.

[0089] The present disclosure also encompasses PTK7-binding proteins that are variants of those specifically described herein. For example, the variants may comprise mutations that confer desired properties to the proteins, e.g., increased half-life, altered (e.g., reduced) immunogenicity in humans, and / or engineered sites for covalent or non-covalent binding toanother molecule (e.g., a cytotoxic, radioactive, or detectable agent). These mutations may be introduced in the framework (FR) regions, the CDRs, or other parts of the proteins (e.g., the Ig constant regions, if the proteins contain such regions).

[0090] In some embodiments, the anti-PTK7 antibody may comprise at least one mutation in the Fc region. A number of different Fc mutations are known, where these mutations alter, e.g., the antibody’s effector functions or half-life. For example, in some embodiments, the constant region of an antibody (e.g., an anti-PTK7 antibody herein) may comprise mutations that improve the therapeutic potential of the antibody, such as mutations that reduce or eliminate effector functions of the antibody. An antibody may comprise, for instance, a human IgGi constant region with the mutation L235E, “LALA” mutations (L234A / L235A), “LALAGA” mutations (L234A / L235A / G237A), “LALAGR” mutations (L234A / L235A / G236R), or “LALAPG” mutations (L234A / L235A / P329G) (Eu numbering is used when referring to constant region mutations, unless otherwise indicated). In some embodiments, the constant region may comprise L234F / L235E / P331S (“FES”), L234F / L235Q / K322Q (“FQQ”), A330S / P331S, L234A / G237A, L234A / L235A / G237A, L234A / L235A / G237A / P238S / H268A / A330S / P330S, L234A / L235E, G236R / L328R, or L234A / L235A / K322A mutations. The present disclosure contemplates, for instance, an anti- PTK7 antibody herein with any mutation that reduces or eliminates effector functions as described in Wilkinson et al., PLoS One (2021) 16(12):e0260954. Additionally or alternatively, the human IgGi constant region may comprise, for instance, a human heavy chain constant region with the mutation P329A. In some embodiments, the monospecific or multispecific antibody herein may comprise a human IgG4 constant region with the mutation L235E and / or the mutation S228P. An IgG constant region may comprise mutations that improve the serum half-life of the antibody and / or improve manufacturing and yield of the antibody.

[0091] In certain embodiments, the anti-PTK7 antibody may comprise, e.g., L234A, L235A, and / or P329A mutations (Eu numbering), wherein the mutations may appear alone or in any combination. In particular embodiments, the anti-PTK7 antibody may comprise an Fc region with all three mutations.

[0092] In some embodiments, the present PTK7 binders, such as anti-PTK7 antibodies, have higher internalization rates on tumor cells as compared to control antibodies such as Hu24. In some embodiments, the binders bind both human PTK7 and cynomolgus PKT7.II. Making of PTK7-Binding Proteins

[0093] The PTK7-binding protein of the present disclosure may be produced recombinantly using isolated nucleic acid molecules such as expression constructs. The coding sequences for each polypeptide chain may be cloned into a single vector or cloned into separate vectors (e.g., a pair or set of vectors). The proteins may be produced in host cells, e.g., mammalian host cells, using appropriate expression constructs. Mammalian cell lines available as hosts for expression include, without limitation, Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH- 3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and A549 cells. Other cell lines that may be used are insect cell lines, such as Sf9 or Sf21 cells, and yeast cell lines. In certain embodiments, the cell lines are not derived from a human embryo. Cell lines may be selected based on their expression levels.

[0094] Host cells used to produce the PTK7-binding proteins are “recombinant host cells.” A “recombinant host cell” (or simply “host cell”), as used herein, means a cell into which a recombinant expression construct has been introduced. By definition, a recombinant host cell does not occur in nature. A protein produced from a recombinant host cell is a recombinant protein.

[0095] The PTK7-binding protein may be isolated and purified from the host cell culture using well known methods, such as centrifugation; ultracentrifugation; protein A, protein G, protein A / G, or protein L purification; and / or ion exchange chromatography.III. Pharmaceutical Compositions

[0096] Another aspect of the present disclosure is a pharmaceutical composition comprising as an active ingredient (or as the sole active ingredient) the PTK7-binding protein of the present disclosure. The pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient. A “pharmaceutically acceptable excipient” may include appropriate solvents, dispersion media, antibacterial and antifungal agents, isotonic agents, and the like. Examples of pharmaceutically acceptable excipients are water and saline (e.g., phosphate-buffered saline).

[0097] The pharmaceutical compositions may be used to treat PTK7-associated diseases, e.g., diseases where PTK7 is overexpressed. In some embodiments, the pharmaceutical compositions are intended for amelioration, prevention, and / or treatment of a PTK7-related disorder and / or cancer. As used herein, a PTK7-related or -mediated disorder refers to adisorder, disease or condition that improves, or slows down in its progression, by modulation of PTK7 activity. In some embodiments, the compositions are intended for activation of the immune system. In certain embodiments, the compositions are intended for amelioration, prevention, and / or treatment of cancer originating in tissues such as skin, lung, intestine, colon, ovary, brain, prostate, kidney, bones, soft tissues, the hematopoietic system, head and neck, liver, bladder, breast, stomach, cervical, endometrial, uterus, gastric, esophageal, and pancreas. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is triple-negative breast cancer (TNBC). In certain embodiments, the cancer is nonsmall-cell lung carcinoma (NSCLC). In certain embodiments, the cancer is small-cell lung carcinoma. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the cancer is cervical cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is endometrial cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is sarcoma. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is esophageal cancer. The cancer may be, e.g., at an early, intermediate, late, locally advanced, or metastatic stage, and may be relapsed or refractory to other therapeutics, or there may be no standard therapy available.

[0098] Treat,” “treating,” and “treatment” refer to a method of alleviating or abrogating a biological disorder and / or at least one of its attendant symptoms. As used herein, to “alleviate” a disease, disorder or condition means reducing the severity and / or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to “treatment” include references to curative, palliative and prophylactic treatment. In some embodiments, therapeutic use of an antibody or antigen-binding portion thereof described herein will result in delayed tumor growth, elimination of cancer cells, tumor shrinkage / regression, increased survival, slowed or decreased metastasis, or other clinical endpoints desired by healthcare professionals. In certain embodiments, therapeutic use of an antibody or antigen-binding portion thereof described herein inhibits tumor growth by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%. In certain embodiments, therapeutic use of an antibody or antigen-binding portion thereof described herein provides partial tumor regression of at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%, or complete tumor regression.

[0099] The PTK7-binding proteins of the present disclosure may be administered without additional therapeutic treatments, i.e., as a stand-alone therapy (monotherapy). Alternatively, treatment with the PTK7 -binding proteins may include at least one additional therapeutic treatment (combination therapy), e.g., an anti-cancer agent (such as a chemotherapeuticagent, an anti-neoplastic agent, or an anti-angiogenic agent), or a vaccine (such as a tumor vaccine).

[0100] The PTK7-binding proteins also are useful in diagnostic processes (e.g., in vitro or ex vivo). For example, the PTK7-binding proteins can be used to detect and / or measure the level of PTK7 in a biological sample from a patient (e.g., a tumor biopsy, a tissue sample, or a blood sample). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistochemistry. The present disclosure further encompasses kits (e.g., diagnostic kits) comprising the antibodies, antigenbinding portions, or binding proteins described herein.

[0101] The pharmaceutical composition may be administered to a patient (e.g., a human) in need thereof in a therapeutically effective amount. A “therapeutically effective amount” is an amount that will relieve to some extent one or more of the symptoms of the disease being treated. A therapeutically effective amount of an anti-cancer therapeutic may, for example, result in delayed cancer growth; elimination of cancer cells; tumor shrinkage; increased survival; prevented, decreased, or slowed metastasis; or other clinical endpoints desired by healthcare professionals.

[0102] The pharmaceutical compositions herein may be delivered to the patient through parenteral administration, e.g., selected from subcutaneous, intraperitoneal, intramuscular, intrasternal, intracisternal, intravenous, intraarterial, intrathecal, intraurethral, intracranial, intratumoral, and intrasynovial injection or infusions. Particular embodiments include the intravenous route (e.g., intravenous infusion) and the subcutaneous route (e.g., subcutaneous injection).

[0103] Pharmaceutical compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington ’s Pharmaceutical Sciences, 19thEdition (Mack Publishing Company, 1995). Pharmaceutical compositions are preferably manufactured under GMP (good manufacturing practices) conditions.

[0104] A pharmaceutical composition of the present disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to asubject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0105] Formulations of a pharmaceutical composition suitable for parenteral administration typically comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In some embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation.

[0106] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of 1integers. All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0107] As used herein, the percent identity of two amino acid sequences (or of two nucleic acid sequences) may be obtained by, e.g., BLAST® using default parameters (available at the U.S. National Library of Medicine’s National Center for Biotechnology Information website). In some embodiments, the length of a query sequence aligned for comparison purposes is at least 30% (e.g., at least 40, 50, 60, 70, 80, or 90%) of the length of the reference sequence.

[0108] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Any compound disclosed herein can be used in any of the treatment methods disclosed herein, wherein the individual to be treated is as defined anywhere herein.

[0109] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.EXAMPLESExample 1: Antibody DiscoveryImmunization

[0110] Four hyperimmune mice (DiversimAb™, Abveris) were immunized with 50 pg of recombinant PTK7 extracellular domain (ECD) protein (ACROBiosystems, cat. #PT7- H52H3). Following the initial immunization, mice were boosted every 2-3 days for 7-10 additional injections. Additionally, five humanized transgenic mice (ATX-GK-mix, Alloy) were immunized with 25 pg recombinant PTK7 ECD using Complete Freund’s adjuvant.Following the initial immunization, mice received 4-7 weekly boosts with 25 pg recombinant PTK7 ECD using Incomplete Freund’s adjuvant.Titer check[oni] Mouse serum titers were checked by ELISA assay. A high binding ELISA plate was coated with 1 pg / mL antigen overnight at 4°C. Both human PTK7-Fc tagged (R&D Systems, cat. #9799-TK-050) and cynomolgus PTK7 his-tagged (ACROBiosystems, cat. #PT7-C52h3) antigen were used. The coating material was aspirated and the wells were blocked with 2% BSA in PBS for Ih at 25°C. The blocking material was removed and 3-fold serial dilutions of sera, beginning at 1 : 100, in blocking solution were added and incubated at 25°C for Ih. The plate was washed four times with PBS containing 0.05% Tween20 (PBST) and HRP-goat anti-mouse IgG, Fcy-specific antibody diluted 1 :20,000 in blocking solution was added and incubated at 25°C for 45 min. The plate was washed five times with PBST and 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was added. The reaction was terminated with stop solution and the plate was read at 450 nm. Normal mouse serum (strain matched) was included as a background control. For comparison, three PTK7 control antibodies were serially diluted 3-fold, beginning at 10 pg / mL.

[0112] The serum titers were also checked for binding to cell lines expressing PTK7 (Jurkat, H520, HCC1428) or not expressing PTK7 (Ramos). Briefly, 100,000 cells were seeded per well, and subsequently, were resuspended in 100 pL of titrated serum, starting at 1 : 100 dilution in 2% BSA in PBS and 3 -fold serially diluted for 11 points. The samples were incubated on ice for 2h, washed, were resuspended in 50 pL of secondary antibody (Alexa Fluor® 488 AffiniPure™ goat anti-mouse IgG, Fey fragment specific, Jackson cat. #115-545- 071) and were incubated for 30 min on ice. The cells were washed and read on an iQue flow cytometer. Normal mouse serum (strain matched) was included as a background control.

[0113] Mice with signals about 10-fold above background on antigen (strain-matched normal serum) at 1 :8100 dilution were used for B cell isolation. Mice that were not used for B cell isolation due to low titers continued to be immunized and serum titers were re-checked 4-5 weeks later.Initial Screening

[0114] Following immunization, B cells were isolated from plasma, spleen and lymph nodes and single B cell screening was performed using the Beacon® Optofluidic System (Berkeley Lights). Using a multiplex assay, cells were screened for IgG secretion, binding torecombinant human PTK7 (extra-cellular domain), binding to recombinant cynomolgus PTK7 (extra-cellular domain), binding to Jurkat cells (express PTK7), and binding to Ramos cells (no PTK7 expression). Isolated B cell clones with different binding profiles were prioritized for antibody sequencing. All selected clones secreted IgG and were negative for binding to Ramos cells. Priority 1 clones were reactive with recombinant human and cynomolgus PTK7 and bound Jurkat cells. Priority 2 clones were reactive with recombinant human and bound Jurkat cells. Priority 3 clones bound Jurkat cells but were unreactive with recombinant PTK7. Complete VH and VL sequences were obtained for 73 antibodies. The antibodies isolated from Beacon® screening are summarized in Table 2. Control anti- huPTK7 antibodies 188B (Miltenyi Biotec cat. #130-091-578; mouse IgG2a), OTI2E7 (Invitrogen cat. #MA5-25774; mouse IgGi) and hu24 (U.S. Pat. No. 9,777,070; humanized murine antibody; VH and VL sequences shown in the sequence table below) are listed at the bottom of Table 2.Table 2. Discovery Campaign AntibodiesExample 2: Secondary Screening and Characterization

[0115] Initially, 48 clones were selected for further characterization and were expressed as human IgGi using Expi293 cells. The antibodies were purified in a single step using MabSelect PrismA™ protein A chromatography resin (Cytiva) and the final buffer composition was 65 mM Tris, 10 mM glycine, pH 6.0.

[0116] Binding of the antibodies to four tumor cell lines expressing PTK7 (Jurkat, NCI- H520, OVCAR3, HCC1428) and one tumor cell line not expressing PTK7 (Ramos) was assessed by flow cytometry. Jurkat is a human T lymphocyte cell line. NCI-H520 is a human non-small cell lung cancer line. OVCAR3 is a human high-grade serous ovarian adenocarcinoma cell line. HCC1428 is a human epithelial adenocarcinoma cell line. Ramos (RA-1) is a human B lymphocyte cell line.

[0117] The binding of all antibodies was tested at a single concentration (10 pg / mL). Briefly, cells were seeded at 100,000 cells per well and were resuspended in 100 pL of purified IgG at 10 pg / mL. The cells were incubated with the antibody for 30 min on ice, washed two times with 100 pL 1% BSA in PBS, washed once with 150 pL 1% BSA and were resuspended in 50 pL of Alexa Fluor® 488 AffiniPure™ goat anti-mouse IgG, Fey fragment specific (Jackson cat. #115-545-071) diluted in 1% BSA in PBS, and were incubated on ice for an additional 30 min. Control anti-PTK7 antibodies 188B, OTI2E7 and hu24 were also tested at 10 pg / mL. The MFI values obtained are summarized in Table 3. In general, NCI-H520 cells displayed the highest MFI values with most antibodies tested while HCC1428 cells displayed the lowest MFI values. However, certain antibodies displayed different staining patterns. For example, control antibody OTI2E7 and several discovered antibodies (e.g., D4D88936-3188, D3D88936-15092, D4D88936-1474, and D4D88750-935) displayed higher binding signals with OVCAR3 cells than with NCI-H520 cells. Several antibodies that displayed strong binding to all four tumor cell lines, but not Ramos cells, wereidentified (e.g., D4D88750-9185, D4D88750-11630, D4D88936-3188, D3D88936-15092, D4D88936-1474, D3D88936-18327, and D4D88750-935). Some antibodies, such as D3D88936-15092, D4D88936-3188, D3D88936-18327 and D4D88750-935, displayed superior binding to tumor cells over control antibody 188B.Table 3. Flow Cytometry Analysis of Binding to Cell Lines

[0118] Twelve additional clones were selected for characterization and were expressed as human IgGi using Expi293 cells. Binding of these antibodies to the four tumor cell lines expressing PTK7 (Jurkat, NCI-H520, OVCAR3, HCC1428) and one tumor cell line not expressing PTK7 (Ramos) was assessed by flow cytometry, as described above. The MFI values obtained are summarized in Table 4. For this set of antibodies, OVCAR3 cells generally displayed the highest MFI values while HCC1428 cells displayed the lowest expression. However, similar to the first panel of antibodies tested, the antibodies displayed different relative binding patterns across the various cell lines tested. Multiple antibodies that displayed strong binding to all four tumor cell lines, but not Ramos, cells were identified (e.g., D2D77808-5632, D2D77808-3707, and D2D77808-7380).Table 4. Flow Cytometry Analysis of Binding to Cell Lines

[0119] Next, the binding kinetics of the antibodies to human and cynomolgus recombinant PTK7 was characterized using the Octet® bio-layer interferometry (BLI) platform (Sartorius). Individual anti-human biosensors were loaded with purified human IgG samples. Following establishment of the loading baseline, the sensors were exposed to human PTK7 (His-tagged, at 250 nM) and the association rates were measured. The biosensors were moved to a buffer well to measure the dissociation rate of target antigen from the immobilized antibody. The biosensors were regenerated to repeat the assay using cynomolgus PTK7. The antibodies displayed a wide range of affinities for recombinant human PTK7 (Table 5; “PF”: poor fit, “NB”: no detectable binding). For example, eleven antibodies had KD <10 nM while eleven had KD > 25 nM.Table 5. Recombinant Human and Cynomolgus PTK7 Binding Kinetics

[0120] The binding kinetics of the second set of antibodies (n = 12) to human and cynomolgus recombinant PTK7 was also characterized using the Octet® BLI platform as described above. The data show that this set of antibodies displayed a wide range of affinities for recombinant human PTK7 (Table 6). For example, five antibodies had KD <1 nM, two antibodies had KD <10 nM, and five had KD > 10 nM.Table 6. Recombinant Human and Cynomolgus PTK7 Binding Kinetics

[0121] The above data show that unlike control antibody OTI2E7, most of the new antibodies identified herein were able to bind to both human and cynomolgus PTK7. This cross-species binding feature is highly advantageous because it would allow these antibodies to be studied in non-human primates in pre-clinical toxicology studies.Example 3: Epitope Binning

[0122] Based on tumor cell binding characteristics, thirty-nine antibodies were selected for characterization by epitope binning. To do this, all thirty-nine purified antibodies were prepared at 10 pg / mL in 10 mM sodium acetate, pH 4.5 buffer and covalently coupled to a Carterra® HC30M chip at duplicate spots using the standard EDC / NHS surface chemistry. Next, 100 nM of human PTK7-His tag was injected over the covalently coupled antibody surfaces followed by the injection of a second mAb (one of the thirty -nine purified antibodies) at 10 pg / mL. After the end of each cycle, the covalently coupled surfaces were regenerated. The entire cycle was repeated until all PTK7 mAbs were injected as the second mAb over the complex of hu PTK7-His / antibody surfaces. The cross-competition data were analyzed using the Carterra’s epitope binning analysis software. The combined dendograms created based on the cross-competition data were utilized to develop community plots.Asymmetries represent mAbs that show different competition profiles based on the order of addition.

[0123] The competition is comprised of four major groupings, which are made up of 17 communities as a result of the uniqueness of the antibodies. The results are summarized in FIG. 1 and in Table 7 (SEQ: SEQ ID NO).Table 7. Epitope Binning of Anti-PTK7 AntibodiesExample 4: Tumor Cell Binding

[0124] The binding of select antibodies to NCI-H520 and Jurkat cells was characterized. Briefly, 250,000 cells were seeded per well in a 96-well plate and were resuspended in 100 pL of staining buffer (2% FBS in RPMI) containing antibody. Antibodies were serially diluted 4-fold, beginning at 30 pg / mL. Samples were incubated on ice for 20 min, washed three times with 250 pL of wash buffer (2% FBS in PBS), resuspended in 100 pL of secondary antibody (goat anti-human Fc PE, Invitrogen cat. #12-4998-82) and wereincubated on ice for 20 min. The cells were washed three times with 250 pL of FACS buffer (2% FBS with 2 mM EDTA in PBS). The cells were either analyzed live or alternatively, were fixed by resuspending in 100 pL of 2% paraformaldehyde in PBS and incubating for 10 min at 25°C. Following fixation, the cells were washed once with 250 pL of FACS buffer and were resuspended in 150 pL of FACS buffer. Control samples included unstained cells, secondary alone, or isotype control. Intact, single cells were isolated and the MFI from the control sample was subtracted.

[0125] The tested antibodies displayed variable binding to tumor cell lines. Specifically, maximal binding to NCI-H520 cells, as indicated by maximum MFI, varied from low (<20 median MFI), to intermediate (20-100 median MFI), to high (>100 median MFI) (FIGs. 2-4). Additionally, the concentration of half-maximal binding (ECso) to NCI-H520 cells varied from 0.3 to >13 nM (Table 8). Likewise, maximal binding and the ECso values of the antibodies’ binding to Jurkat cells was also variable (FIG. 5 and Table 8).Table 8. Antibody Binding to Tumor Cell Lines*N.D. indicates low binding or non-saturable binding at the concentrations tested.

[0126] The above data show that control antibody Hu24 exhibited nanomolar or sub- nanomolar EC50, whereas some of the new antibodies, such as D3D88936-18327 and D2D77808-3188, exhibited EC50 values that, while in the nanomolar range, were several -fold higher than that of Hu24. This finding is consistent with data from the soluble PTK7 binding assay, in which these two new antibodies showed KD values that were one order of magnitude higher than that of Hu24 (Tables 5 and 6). The range of PTK7-binding affinity displayed by these new antibodies would be advantageous over Hu24 in two aspects: (i) lower binding affinity for soluble PTK7 would diminish antigen sink effects, (antibody bound by soluble PTK7 en route to tumor sites reduces the antibody’s tumor-targeting efficiency); and (ii) slightly lower antigen-binding affinity on tumor cells would reduce tumor binding-site barrier effects, (peripheral tumor cells bind tightly to high affinity antibody, reducing penetration deep into the tumor tissue by the antibody).Example 5: Antibody Internalization

[0127] The internalization of select antibodies on NCI-H520 cells was characterized. Briefly, 30 pg / mL antibody was incubated with 5 x 105cells per sample on ice for 20 min with occasional gentle mixing. Unbound antibody was removed with three washes using ice- cold assay buffer (2% FBS in RPMI). Following the last wash, the cells were resuspended in ice-cold assay buffer and were transferred to a 37°C water bath for the indicated time. To terminate internalization, the cells were pelleted at 500g for 3 min at 4°C, the supernatant was aspirated, and the cells were resuspended in 100 pL of secondary antibody (goat anti-human Fc PE, Invitrogen cat. #12-4998-82) and were incubated on ice for 20 min. Samples were washed three times with 250 pL of ice-cold 2% FBS, 2 mM EDTA in PBS. For analysis of live cells, the cells were resuspended in 150 pL of 2% FBS, 2 mM EDTA in PBS and were analyzed by flow cytometry. Alternatively, after the final wash the cells were fixed in 100 pL of paraformaldehyde in PBS for 10 min at 25°C. The cells were then washed once with 250 pL of 2% FBS, 2 mM EDTA in PBS, resuspended in 150 pL of 2% FBS, 2 mM EDTA in PBS and were analyzed by flow cytometry. The binding signal obtained from cells incubated on ice the entire time (time 0) was designated 100% surface binding and internalization was quantitated by measuring the loss of binding signal over time following incubation at 37°C.

[0128] Antibodies displaying a broad range of internalization were identified. The decrease in surface bound antibody was least for SLX-1035 (12%) and SLX-1071 (15%), while SLX-1040 (80%) and SLX-1049 (91%) displayed the greatest decrease in surface bound antibody at 3 hrs (FIG. 6). Multiple antibodies displayed intermediate decreases in surface binding, ranging from 40 - 62% at 3 hrs (FIGs. 6-8). In general, antibodies from community 3 of the epitope binning experiment (FIG. 1), or the closely-related community 11, displayed the greatest internalization activity. Antibodies from community 2, including the benchmark SLX-1000, also displayed good internalization activity (30 - 60% decrease in surface binding). Notably, some of the new antibodies, such as SLX-1040 (i.e., D3D88936- 18327) and SLX-1049 (i.e., D4D88750-11630), displayed higher levels of internalization as compared to control antibody SLX-1000 (i.e., Hu24) (FIG. 6). Antibodies with high internalization rates would be advantageous for serving as tumor-targeting moieties in antibody-drug conjugates.Example 6: Sequence Analysis, Template Selection, Framework Mutations

[0129] The variable region sequences of murine antibody D3D88936-18327 (SLX-1040) heavy (SEQ ID NO:76) and light chains (SEQ ID NO:75) were analyzed for potential post- translational modification sites and the CDRs were identified. Multiple heavy and light chain CDR definitions were determined, as shown in FIG. 9A. For CDR grafting, the CDRs were defined as the combination of Kabat, Chothia and IMGT definitions. For humanization, the final rows in the tables of FIG. 9A (Grafted Seq) indicate the residues that were grafted into human germline frameworks.

[0130] The most homologous human germline framework sequences were identified to be used as acceptor sequences for humanization. The sequence alignment of the heavy and light chains of SLX-1040 with the most homologous human germline heavy chain, IGHV1- 46*01, and light chain, IGKV1D-16*O1, is shown in FIG. 9B. Certain framework residues were identified as being potentially important for preserving the binding activity of SLX- 1040 and these residues are marked with an asterisk in FIG. 9B. Multiple variants containing various combinations of back-mutations (human germline framework residues mutated back to corresponding murine framework residue) were synthesized (Z1 through Z22; FIG. 9C) and expressed as IgGi. A chimeric version of SLX-1040 was also made, with the murine VH and VL, and human IgGi and K constant regions (chi 8327). The framework (FR) back mutations and heavy and light chain combinations of the variants tested are summarized inFIG. 9C. The variants were expressed transiently in Expi293F™ cells and were purified using Protein A chromatography.Example 7: Characterization of Humanized Variants

[0131] The humanized variants were characterized for binding to recombinant human and cynomolgus PTK7, binding to a human tumor cell line expressing PTK7 (NCI-H520), and binding to recombinant CHOK1 cell line transfected with cynomolgus PTK7.

[0132] Binding of the purified variants to the extracellular domain (ECD) of recombinant human PTK7 (ACROBiosystems, cat. #PT7-H52H3) was characterized by SPR using a Biacore™ 8K. Antibody was captured using anti-human Fc IgG (Jackson ImmunoResearch) immobilized on a CM5 chip (Cytiva). The antibodies were diluted 100- to 200-fold and the contact time was 60 s at a flow rate of 10 pL / min. Recombinant PTK7 was used at a working concentration of 500 nM, with 180 s association time and a 600 s dissociation time at a flow rate of 30 pL / min. The running buffer was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4. The binding data was evaluated using a 1 : 1 binding model and Biacore™ evaluation software, using the following formula:Relative Binding Response (%) = 100 x [Binding Response (PTK7) x MW (mAb)] / [Capture Level (mAb) x MW (PTK7) x 2]

[0133] Based on analyzing the dissociation rate, binding response, and relative binding response the most active variants containing the fewest back mutations were Z10 - Z15 (Table 9)Table 9. Binding of chl8327 and Humanized Variants to Human PTK7 ECD

[0134] Next, the binding of the purified variants to the extracellular domain (ECD) of recombinant cynomolgus PTK7 (ACROBiosystems, cat. #PT7-C52h3) was characterized by SPR using a Biacore™ 8K. Antibody was captured using anti-human Fc IgG (Jackson ImmunoResearch) immobilized on a CM5 chip (Cytiva). The antibodies were diluted 10- to 50-fold and the contact time was 60 s at a flow rate of 10 pL / min. Recombinant PTK7 was used at a working concentration of 500 nM, with 180 s association time and a 300 s dissociation time at a flow rate of 30 pL / min. The running buffer was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4 (Cytiva, Cat. BR100669). The binding data was evaluated using a 1 : 1 binding model and Biacore™ evaluation software as described above.

[0135] Based on analyzing the dissociation rate, binding response, and relative binding response the most active variants containing the fewest back mutations were Z12 - Z14 (Table 10)Table 10. Binding of chl8327 and Humanized Variants to Cynomolgus PTK7 ECD

[0136] The binding of the variants to the human tumor cell line NCI-H520 was characterized using flow cytometry. Briefly, five-fold serial dilutions of antibody were incubated with IxlO5cells for 1 h at 4°C. Unbound antibody was removed by washing and the cells were incubated with anti-human IgG-Fc-PE diluted 100-fold for 30 min at 4°C. Unbound antibody was removed by washing and the cells were analyzed by flow cytometry. Representative binding of some of the variants is shown in FIG. 10. Variant Z1 (FIG. 10, open squares) displayed significantly diminished binding, while variants Z4 (open triangles) and Z7 (open inverted triangles) displayed slightly diminished binding. However, multiple variants display similar maximum MFI and cell binding ECso (FIG. 10, compare Z 10, Z12, Z22 with chimeric 18327). No binding was observed with an isotype control antibody (FIG. 10, inverted closed triangles). The maximum MFI and ECso (nM) for binding for all the humanized variants are summarized in Table 11.Table 11. Binding of chl8327 and Humanized Variants to NCI-H520 and CHO cells Expressing Cynomolgus PTK7

[0137] Next, the binding of the variants to CHO cells expressing cynomolgus PTK7 was characterized using flow cytometry. Briefly, five-fold serial dilutions of antibody were incubated with IxlO5cells for 1 h at 4°C. Unbound antibody was removed by washing and the cells were incubated with anti-human IgG-Fc-PE diluted 100-fold for 30 min at 4°C. Unbound antibody was removed by washing and the cells were analyzed by flow cytometry. Representative binding is shown in FIG. 11. Variant Z1 (open squares) displayed minimal binding to CHO cells expressing cynomolgus PTK7. Multiple variants, including variant Z22 (FIG. 11, closed triangles) displayed a similar maximum MFI to chimeric 18327 (FIG. 11, open circles). Similarly, multiple variants bound the transfected CHO cells with cell binding ECso comparable to chimeric 18327 (FIG. 11, compare Z7, Z10, Z12, and Z22 with chimeric 18327). No binding was observed with an isotype control antibody (FIG. 11, inverted closed triangles). The maximum MFI and EC50 (nM) for binding to CHO cells transfected with cynomolgus PTK7 are summarized in Table 11.

[0138] Based on the binding of humanized variants to recombinant human and cynomolgus PTK7 ECD and on binding to cells expressing human or cynomolgus PTK7 certain murine framework residues were identified as potentially important for preserving the activity of the murine antibody 18327. Specifically, variant Z 12 displayed the best overall binding to recombinant human and cynomolgus PTK7 and to cells expressing human and cynomolgus PTK7, while containing the fewest back mutations of the variants expressed inthis first panel. Variant Z12 contains Y91F, R71 V, V78A back mutations in the VH and Y36L, S46R, G66R, F71 Y mutations in the VL.Example 8: Further Refinement of Humanized Variants

[0139] In an attempt to further reduce the total number of back mutations while preserving the binding activity of the antibody, twelve additional variants (Z23 - Z34) were synthesized and characterized (FIG. 9D). The variants were expressed transiently in Expi293F™ cells and were purified using Protein A chromatography. The variants all expressed well and were purified to >99% as assessed by analytical SEC, with the exception of variants Z27 and Z32, which expressed well and were purified to >93% as assessed by analytical SEC.

[0140] The humanized variants were characterized for binding to recombinant human and cynomolgus PTK7, binding to a human tumor cell line expressing PTK7 (NCI-H520), and binding to recombinant CHOK1 cell line transfected with cynomolgus PTK7.

[0141] The binding of the purified variants to the ECD of recombinant human PTK7 and the ECD of recombinant cynomolgus PTK7 was characterized by SPR using a Biacore™ 8K. Antibody was captured using anti-human Fc IgG (Jackson ImmunoResearch) immobilized on a CM5 chip (Cytiva). The antibodies were diluted to 1.5 pg / mL and were captured for 80 s at a flow rate of 10 pL / min. Recombinant human PTK7 was used at a working concentration of 500 nM, with 180 s association time and a 600 s dissociation time at a flow rate of 30 pL / min, while recombinant cynomolgus PTK7 was used at a working concentration of 500 nM, with 180 s association time and a 300 s dissociation time at a flow rate of 30 pL / min.The running buffer was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4. The binding data was evaluated using a 1 : 1 binding model and Biacore™ evaluation software as described above.

[0142] The variants all displayed similar dissociation rates (ka) with human recombinant PTK7, ranging from 1.34xl0'3s'1(Z12) to 3.90x'3s'1(Z27), as summarized in Table 12. The dissociation rates of the humanized variants were slightly faster than the chimericl8327 (6.96xl0‘4s'1). Four variants had ka rates within 2-fold of the chimeric 18327: Z12, Z23, Z29, and Z31. Additionally, the Relative Binding Response of the humanized variants ranged from 33% (Z26) on the low end to 43% (Z32) on the high end (Table 12). The Relative Binding Response of variants Z23 (40%) and Z32 (43%) were closest to chimeric 18327 (50%).Table 12. Binding of chl8327 and Humanized Variants to Human PTK7 ECD

[0143] The variants all displayed similar dissociation rates at 300 s with cynomolgus recombinant PTK7, ranging from 3.08xl0'3s'1(Z27) to 8.67xl0'3s'1(Z29), as summarized in Table 13. The dissociation rates of the humanized variants were similar to chimeric 18327 (6.97xl0‘3s'1), with chimeric 18327 dissociating approximately ten times faster from cynomolgus PTK7 than from human PTK7. For binding to recombinant cynomolgus PTK7 the antibodies showed heterogeneous binding, with faster off rates observed in the first 120 s followed by very slow dissociation. Consequently, the dissociation rates at 120 s were also characterized. Again, the variants were similar, displaying dissociation rates ranging from 1.30xl0'3s'1(Z25) to 9.66xl0'3s'1(Z32). Dissociation of the variants at 120 s was equivalent or slightly faster than was observed with chimeric 18327 at 120 s (8.71xl0‘3s'1), with the exception of Z27 (7.60xl0‘3s'1). The Relative Binding Response of the humanized variants ranged from 8% (Z26) on the low end to 16% (Z32) on the high end (Table 13). The Relative Binding Response of multiple variants was 12 -16%, compared to 24% for chimeric 18327. In general, the binding of chimeric 18327 and humanized variants to recombinant cynomolgus PTK7 was weaker than their binding to recombinant human PTK7.Table 13. Binding of chl8327 and Humanized Variants to Cynomolgus PTK7 ECD

[0144] The binding of the variants to the human tumor cell line NCI-H520 and CHO cells expressing cynomolgus PTK7 was characterized using flow cytometry, as described in Example 7. Multiple variants bound NCI-H520 with an ECso and maximum MFI similar to chimeric 18327. For example, the binding profiles of humanized clones Z12, Z23, Z29, Z31, and Z32 were all similar to chimeric 18327 (FIG. 12). Likewise, multiple variants bound CHO cells transfected with cynomolgus PTK7 similar to chimeric 18327 (FIG. 13). The maximum MFI and ECso (nM) for binding to both cell lines are summarized in Table 14.Table 14. Binding of chl8327 and Humanized Variants to NCI-H520 and CHO cells Expressing Cynomolgus PTK7Example 9: Thermal Stability of Select Humanized Variants

[0145] The thermal stability of select humanized variants was characterized by Differential Scanning Fluorimetry (DSF). Chimeric 18327 and variants Z12, Z23, Z29, and Z31 were suspended in PBS at 1 mg / mL and the Tmi(°C) was measured twice. The variants mean Tmiranged from 68.4°C (Z29) to 71.2°C (Z23), all higher than chimeric 18327 Tmi(64.5°C), as summarized in Table 15.Table 15. Thermal Stability of chl8327 and Select Humanized VariantsExample 10: Binding Kinetics of Select Humanized Variants

[0146] The binding of the purified variants to the ECD of recombinant human PTK7 was characterized by SPR using a Biacore™ 8K. Antibody was captured using anti-human Fc IgG (Jackson ImmunoResearch) immobilized on a CM5 chip (Cytiva). The antibodies (ligand) were diluted to 1.5 pg / mL and were captured for 70 s at a flow rate of 10 pl / min. Recombinant human PTK7 (analyte) was used at seven concentrations, beginning at 500 nM and diluted serially, two-fold to 7.813 nM with 180 s association time and a 600 s dissociation time at a flow rate of 30 pL / min. The running buffer was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4. The binding data was evaluated using a 1 : 1 binding model and Biacore™ evaluation software. The humanized antibodies all displayed binding affinities similar, though slightly less than, chimeric 18327. The results are summarized in Table 16.Table 16. Antibody Binding Kinetics to Human PTK7

[0147] Next, the binding of the purified variants to the ECD of recombinant cynomolgus PTK7 was characterized by SPR using a Biacore™ 8K. Antibody was captured using antihuman Fc IgG (Jackson ImmunoResearch) immobilized on a CM5 chip (Cytiva). The antibodies (ligand) were diluted to 2 pg / mL (chimeric 18327) or 3 pg / mL (humanized variants) and were captured for 70 s at a flow rate of 10 pL / min. Recombinant PTK7 (analyte) was used at seven concentrations, beginning at 500 nM and diluted serially, twofold to 7.813 nM. With 180 s association time and a 300 s dissociation time at a flow rate of 30 pL / min. The running buffer was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Tween 20, pH 7.4. The binding data was evaluated using a 1 : 1 binding model and Biacore™ evaluation software. The humanized antibodies all displayed binding affinities similar, though slightly less than, chimeric 18327. The results are summarized in Table 17.Table 17. Kinetics of Antibody Binding to Cynomolgus PTK7Example 11: Binding of Humanized Variants to NCI-H520 (human) and CMMT (rhesus) Cell Lines

[0148] The binding of a subset of humanized variants to human (NCI-H520) and rhesus(CMMT) tumor cell lines was investigated using flow cytometry. The protein sequence of the extracellular domain of rhesus and cynomolgus PTK7 is identical. Briefly, log-phase growing cells were detached using Accutase® solution (Millipore Sigma) at 37°C, diluted into culture media and collected by centrifugation at 500 x g for 3 min. The cells were resuspended in cold 2% FBS in RPMI (binding buffer) at 2 x 106cells / mL. Next, cells were seeded at 50 pL per well (100,000 cells) in a 96-well v-bottom plate. Antibodies were serially diluted 4-fold in binding, beginning at 100 pg / mL, and 50 pL per well was added to the cells and incubated on ice for 30 min. The samples were washed three times with 200 pL of 2% FBS in PBS (FACS buffer), resuspended in 100 pL of secondary antibody (goat antihuman Fc PE, Invitrogen cat. #12-4998-82) diluted 500-fold in binding buffer and were incubated on ice (in the dark) for 30 min. The secondary antibody solution also contained Fixable Viability Dye eFluor™ 780 (Invitrogen) diluted 200-fold. The cells were washed three times with 200 pL of FACS buffer and were fixed with 100 pL of 2% paraformaldehyde aqueous solution (PF A) (Electron Microscopy Sciences) at 25°C (in the dark) for 10 min. The cells were washed two times with 200 pL of FACS buffer, resuspended in 200 pL of 2 mM EDTA in PBS, and were stored at 4°C in the dark until analyzed by flow cytometry. Antibody binding was quantitated by measuring the PE median fluorescence intensity (MFI) from 10,000 events of viable singlet cells. Intact, single cells were isolated and the MFI from the control sample (secondary antibody only) was subtracted.

[0149] As expected, the binding of the humanized variants to NCI-H520 cells was very similar to the binding of chimeric 18327 (FIG. 14, Table 18). The maximum MFI of the variants was indistinguishable from that of chimeric 18327, while the ECsos were all within a 1.5-fold range of one another (3.8 nM - 5.5 nM). The binding of the humanized variants to CMMT cells was more variable with some variants displaying a lower maximum MFI than others (FIG. 15, Table 19, compare Z12 versus Z29).Table 18. ECso Values of Humanized Variants’ Binding to NCI-H520 CellsTable 19. ECso Values of Humanized Variants’ Binding to CMMT Cells

[0150] The ECsos for binding to CMMT cells were all within a 1.5-fold range of one another, as was observed with binding to the human tumor cell line NCI-H520. Interestingly, although the binding of the humanized variants to recombinant cynomolgus PTK7 was weaker than their binding to recombinant human PTK7 (see Example 9), binding to the cynomolgus tumor cell line CMMT was approximately 2-fold stronger than binding to the human tumor cell line NCI-H520.Example 12: Internalization of Humanized Variants on NCI-H520 Cells

[0151] The internalization of the chimeric antibody and various humanized constructs on the non-small cell lung carcinoma NCI-H520 tumor cell line was evaluated. Log-phasegrowing cells were detached using Accutase® (Millipore Sigma) at 37°C, diluted into culture media and collected by centrifugation at 500 x g for 3 min. The cells were resuspended in cold 2% FBS in RPMI (binding buffer) at 2 x 106cells / mL. Next, cells were incubated with 30 pg / mL antibody on ice for 30 min. Unbound antibody was removed with three washes using ice-cold 2% FBS in PBS (FACS buffer). Following the last wash, the cells were resuspended in 1 mL ice-cold binding buffer and 100 pL (200,000 cells) was aliquoted for each time point of the internalization time course. The samples were incubated in a water bath at 37°C for the indicated time. To terminate internalization, the cells were transferred to ice. Subsequently, the cells were collected by centrifugation at 500 x g for 3 min at 4°C, the supernatant was aspirated, and the cells were resuspended in 100 pL of secondary antibody (goat anti-human Fc PE, Invitrogen cat. #12-4998-82) diluted 500-fold in binding buffer and were incubated on ice for 30 min. The secondary antibody solution also contained Fixable Viability Dye eFluor™ 780 (Invitrogen) diluted 200-fold. Samples were washed three times with 200 pL of ice-cold FACS buffer and were fixed with 100 pL of 2% paraformaldehyde aqueous solution (PF A) (Electron Microscopy Sciences) at 25°C (in the dark) for 10 min. The cells were washed two times with 200 pL of FACS buffer, resuspended in 200 pL of 2 mM EDTA in PBS, and were stored at 4°C in the dark until analyzed by flow cytometry. Antibody binding was quantitated by measuring the PE median fluorescence intensity (MFI) from 10,000 events of viable singlet cells. The binding signal obtained from cells incubated on ice the entire time (time 0) was designated 100% surface binding and internalization was quantitated by measuring the loss of binding signal (MFI) over time following incubation at 37°C.

[0152] The humanized variants all internalized as well, or better than, chimeric 18327 (FIG. 16), demonstrating that this antibody characteristic was maintained through the humanization process.Example 13: Developability Studies

[0153] The developability of certain humanized variants was characterized by evaluating their serum stability, colloidal stability, stability to freeze / thaw cycles, hydrophobicity, and their response to thermal stress.

[0154] In order to assess serum stability, the antibodies were incubated in human serum at 37°C for 14 days. Subsequently the antibody binding to antigen was characterized by ELISA. Briefly, recombinant human PTK7 was incubated at 1 pg / ml on a microtiter plate at 4°C overnight. The plate was then blocked with 200 pL / well with 2% BSA in PBS for Ih at25°C. Next antibody was diluted serially 5-fold, beginning at 300 nM, and incubated for 2h at 25°C. Antibody binding was detected with goat anti-human IgG-Fc-HRP diluted 1 :5000 and quantitated by incubating with TMB substrate. The reaction was terminated with the addition of IM HC1 and read at OD450 on a microtiter plate reader. The maximum OD450 and EC50 were calculated. No appreciable change in maximum (Max.) OD450 nor in EC50 was detected over 14 days for Z12, Z23 and Z31, as summarized in Table 20.Table 20. Serum Stability

[0155] Next, the propensity of the humanized variants to associate because of weak attractive forces associated with hydrophobic surface residues and / or surface charges was assessed using dynamic light scattering (DLS). DLS was performed using DynaPro® Plate Reader III. Antibodies Z12, Z23 and Z31were suspended to 2.5, 5, 10, 15 and 20 mg / mL in 45 mM sodium citrate, 135 mM NaCl, 100 mM arginine, 40 mM succinic acid, pH 5.5, transferred into a 1536-well microplate, and five scans of 5s each were accumulated for each sample. All samples were measured at 25°C in duplicate wells. The diffusion coefficient was determined using DYNAMICS operation software (v7.8.1.3) and was further plotted against protein concentration to obtain the Diffusion interaction parameter (fo) value. As summarized in Table 21, theof all three humanized variants was > -10 (mL / g), consistent with monodisperse size (low propensity to aggregate).Table 21. DLS Characterization

[0156] The stability of certain humanized variants subjected to five freeze (-80°C) / thaw (25°C) cycles was assessed. Variants Z12, Z23, and Z31 were resuspended at 1 mg / mL in 45 mM sodium citrate, 135 mM NaCl, 100 mM arginine, 40 mM succinic acid, pH 5.5. The protein concentration, purity by SEC-HPLC, and charge distribution by iCIEF were measured before freezing and after five freeze / thaw cycles. No obvious changes in protein concentration, purity and charge distribution were observed for any of the variants, as summarized in FIG. 17.

[0157] The relative hydrophobicity of chimeric 18327, Z12, Z23, and Z31 were compared using HIC-HPLC. The variants were resuspended at 1 mg / mL in 45 mM sodium citrate, 135 mM NaCl, 100 mM arginine, 40 mM succinic acid, pH 5.5 and were separated on a Tosoh TSKgel® butyl-NPR (4.6 mm I.D. x 10 cm, 2 pm) column using a gradient (flow rate of 0.5 mL / min at 25°C), as outlined in Table 22. Mobile phase solution A was 25 mM phosphate buffer, pH 7.0 and solution B was 1.5 M (NH^SCU, 25 mM phosphate buffer, pH 7.0. Z12, Z13 and Z31 all displayed similar retention times (44.6, 44.4, and 44.8 min, respectively), which were longer than chimeric 18327 (36.1 min).Table 22. HIC-HPLC Mobile Phase Gradient Conditions

[0158] The thermal stability of certain humanized variants incubated for 14 days at either 4°C or 40°C was characterized. Variants Z12, Z23, and Z31 were resuspended at 1 mg / mL in 45 mM sodium citrate, 135 mM NaCl, 100 mM arginine, 40 mM succinic acid, pH 5.5. The protein concentration, purity by SEC-HPLC, and charge distribution by iCIEF were measured following the 14-day incubation. No obvious changes in protein concentration were observed following incubation for 14 days at 40°C versus 4°C (FIG. 18). There was a small decrease in the monomeric peak and a corresponding small increase in the low molecular weight peak (LMW) in the samples incubated at 40°C, as determined by SEC-HPLC (FIG. 18). With respect to charge variants, Z12, Z23 and Z31 all displayed a decrease in the main peak (with a corresponding increase in the acidic peak content) after the 14-day incubation at 40°C(FIG. 18)SEQUENCES

[0159] The table below shows sequences described herein (SEQ: SEQ ID NO).

Claims

CLAIMS1. An isolated anti-PTK7 antibody or an antigen-binding portion thereof, comprising heavy chain CDR (HCDR) 1-3 and light chain CDR (LCDR) 1-3 comprising SEQ ID N0s: 151, 157, 161, 164, 167, and 170, respectively.

2. The isolated antibody or portion of claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising SEQ ID NOs:76 and 75, respectively.

3. The isolated antibody or portion of claim 1, comprising a VH comprising SEQ ID NOs: 154, 155, and 161, and a VL comprising SEQ ID NOs: 163, 166, and 170.

4. The isolated antibody or portion of claim 3, wherein the antibody or portion is humanized.

5. The isolated antibody or portion of claim 4, wherein the VH comprises framework regions derived from a human IGHV1 -46*01 germline gene.

6. The isolated antibody or portion of claim 5, wherein the VH comprises one or more back mutations selected from Y91F, R71 V, V78A, M48I, M69L, and V20L, wherein the numbering is according to SEQ ID NO: 172.

7. The isolated antibody or portion of claim 4, wherein the VL comprises framework regions derived from a human IGKV1D-16*O1 germline gene.

8. The isolated antibody or portion of claim 7, wherein the VL comprises one or more back mutations selected from I21L, Y36L, A43T, S46R, G66R, T69S, and F71 Y, wherein the numbering is according to SEQ ID NO: 183.

9. The isolated antibody or portion of claim 4, wherein a) the VH comprises any one of SEQ ID NOs: 172-182, 193, 196, and 197, or an amino acid sequence at least 95% identical thereto;b) the VL comprises any one of SEQ ID NOs: 183-192, 194, and 195, or an amino acid sequence at least 95% identical thereto; or c) a) and b).

10. The isolated antibody or portion of claim 9, wherein the VH and the VL compriseSEQ ID NOs: 178 and 188, respectively,SEQ ID NOs: 193 and 188, respectively, or SEQ ID NOs: 173 and 188, respectively.

11. The isolated antibody or portion of any one of the preceding claims, wherein the antibody comprises a human IgGi constant region.

12. The isolated antibody or portion of claim 11, wherein the human IgGi constant region comprises SEQ ID NO:201, optionally without the C-terminal lysine.

13. The isolated antibody or portion of any one of the preceding claims, wherein the antibody comprises a human light chain constant region that comprises SEQ ID NO: 199.

14. An isolated anti-PTK7 antibody comprising a heavy chain that comprises the amino acid sequences of SEQ ID NOs: 193 and 201, optionally without the C-terminal lysine, and a light chain that comprises the amino acid sequences of SEQ ID NOs: 188 and 199.

15. An isolated anti-PK7 antibody comprising a) HCDR1-3 and LCDR1-3 comprising SEQ ID NOs:204, 209, 213, 216, 219, and 222, respectively; b) VH and VL comprising SEQ ID NOs: 112 and 111, respectively; or c) an HC comprising SEQ ID NOs: 112 and 201, optionally without the C-terminal lysine, and an LC comprising SEQ ID NOs: 111 and 199.

16. An isolated anti-PK7 antibody comprising a) HCDR1-3 and LCDR1-3 comprising SEQ ID NOs:226, 231, 235, 238, 241, and 244, respectively; b) VH and VL comprising SEQ ID NOs: 128 and 127, respectively; orc) an HC comprising SEQ ID NOs: 128 and 201, optionally without the C-terminal lysine, and an LC comprising SEQ ID NOs: 127 and 199.

17. The isolated antibody or portion of any one of the preceding claims, wherein the antibody or portion is conjugated to a cytotoxin or a detectable label.

18. A pharmaceutical composition comprising the isolated antibody or portion of any one of claims 1-17 and a pharmaceutically acceptable excipient.

19. Nucleic acid molecule(s) encoding the isolated antibody or portion of any one of the claims 1-17.

20. Expression vector(s) comprising the nucleic acid molecule(s) of claim 19.

21. A recombinant host cell comprising the expression vector(s) of claim 20.

22. A method of making an anti-PTK7 antibody or an antigen-binding portion thereof, comprising: culturing the recombinant host cell of claim 21 under conditions that allow expression of the antibody or portion, and isolating the antibody or portion from the cell culture.

23. A method of treating a PTK7-positive cancer in a patient in need thereof, comprising administering to the patient the isolated antibody or portion of any one of claims 1-17 or the pharmaceutical composition of claim 18.

24. Use of the antibody or portion of any one of claims 1-17 for the manufacture of a medicament for treating a PTK7-positive cancer in a patient in need thereof.

25. The antibody or portion of any one of claims 1-17, or the pharmaceutical composition of claim 18, for use in treating a PTK7-positive cancer in a patient in need thereof.

26. The method of claim 23, the use of claim 24, or the antibody or portion for use of claim 25, wherein the PTK7-positive cancer is selected from head and neck cancer, non-smallcell lung cancer, small-cell lung cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, and sarcoma.

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