Human CDCP1 binding molecules for treating cancer

Abstract

Provided herein are human CDCP1 binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human CDCP1 binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) having particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat cancer (e.g., colon, bladder, and breast cancer). In some embodiments, the CDCP1 binding molecules are conjugated to a cytotoxin (e.g., via a cleavable linker) so as to allow the specific targeting and delivery of a cytotoxic payload to selectively kill cancerous cells.

Description

[0001] Attorney Docket Number: CCF-44473.601

[0002] HUMAN CDCP1 BINDING MOLECULES FOR TREATING CANCER

[0003] The present application claims priority to US Provisional application serial number 63 / 747,183, filed January 20, 2025, which is herein incorporated by reference in its entirety.

[0004] This invention was made with government support under EY025373, EY030111, and EY031463 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] Provided herein are human CDCP1 binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human CDCP1 binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) having particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat cancer (e.g., colon, bladder, and breast cancer). In some embodiments, the CDCP1 binding molecules are conjugated to a cytotoxin (e.g., via a cleavable linker) so as to allow the specific targeting and delivery of a cytotoxic payload to selectively kill cancerous cells.

[0007] BACKGROUND OF THE INVENTION

[0008] Cancer is a major health concern for many individuals.

[0009] SUMMARY OF THE INVENTION

[0010] Provided herein are human CDCP1 binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human CDCP1 binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) having particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat cancer (e.g., colon, bladder, and breast cancer). In some embodiments, the CDCP1 binding molecules are conjugated to a cytotoxin (e.g., via a cleavable linker) so as to allow the specific targeting and delivery of a cytotoxic payload to selectively kill cancerous cells.Attorney Docket Number: CCF-44473.601

[0011] DESCRIPTION OF THE FIGURES

[0012] Figure 1. Characterization of novel anti-CDCPl mAbs. Different developed antihuman CDCP1 mAbs were used to stain WT and CDCP1 KO MDA468 cells then analyzed in flow cytometric assays, showing that these 9A2 mAbs specifically binds to CDCP1 on the cell surface.

[0013] Figure 2. CDCP1 is expressed at high levels on all the bladder cancer cell lines examined. Different established bladder cancer cell lines were stained with 9A2 (blue peaks) or the same concentration of isotype control (pink peaks), then analyzed in flow cytometric assays, showing that all cell lines examined express CDCP1 at high levels.

[0014] Figure 3. CDCP1-ADC kills bladder cancer cells in vitro. The bladder cancer cells (SW780) were incubated with different concentrations of CDCP1-ADC or non-binding ADC (IgG-ADC, control) for 72 hrs and cell viabilities were assessed by a commercial kit, showing that the CDCP1-ADC but not the control ADC, kills the bladder cancer cells in a concentration-dependent manner.

[0015] Figure 4. In Vivo Efficacy. Mouse orthotopic bladder carcinoma model: SW780-luc human bladder carcinoma cells ( 10,000 cells in 50 uL) were inoculated into bladders of female NSG mice via urethral catheterization. Mice received 20 ug ADC intravesically on dl, d4, dl 1 (in 50 uL water), and on dl8 and d26 (in 200 uL DMEM).

[0016] Figure 5A shows the mAb 9A2 heavy chain amino acid sequence (SEQ ID NO: 1) with added leader sequence in italics, and Figure 5B shows the mAb 9A2 light chain amino acid sequence (SEQ ID NO:2) with added leader sequence in italics.

[0017] Figure 6A shows the mAb 9A2 variable region heavy chain amino acid sequence (SEQ ID NOG), including CDRII1 (SEQ ID NO:4), CDRII2, (SEQ ID NOG), and CDRII3 (SEQ ID NO:6). Figure 6B shows the mAb 9A2 variable light chain amino acid sequence (SEQ ID NOG), including CDRL1 (SEQ ID NOG), CDRL2, (SEQ ID NO:9), and CDRL3 (SEQ ID NOTO).

[0018] DEFINITIONS

[0019] To facilitate an understanding of the invention, a number of terms are defined below. The term "antibody," as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each lightAttorney Docket Number: CCF-44473.601

[0020] chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each variable region (VH or VL) contains 3 CDRs, designated CDR1, CDR2 and CDR3 (see, Figure 6). Each variable region also contains 4 framework sub-regions, designated FR 1 , FR2, FR3 and FR4, which may be human framework sub-regions.

[0021] As used herein, the term "antibody fragment or portion" refers to a portion of an intact antibody. Examples of antibody fragments or portions include, but are not limited to, linear antibodies, single-chain antibody molecules, Fv, Fab and F(ab')2 fragments, and multispecific antibodies formed from antibody fragments. The antibody fragments preferably retain at least part of the heavy and / or light chain variable region.

[0022] As used herein, the terms "complementarity determining region" and "CDR" refer to the regions that are primarily responsible for antigen-binding. There are three CDRs in a light chain variable region (CDRL1, CDRL2, and CDRL3), and three CDRs in a heavy chain variable region (CDRH1, CDRH2, and CDRH3).

[0023] As used herein, the term "framework" refers to the residues of the variable region other than the CDR residues. There are four separate framework sub-regions that make up the framework: FR1, FR2, FR3, and FR4. In order to indicate if the framework sub-region is in the light or heavy chain variable region, an "L" or "H" may be added to the sub-region abbreviation (e.g., "FRL1" indicates framework sub-region 1 of the light chain variable region). It is noted that, in certain embodiments, the human CDCP1 binding molecules of the present invention may have less than a complete framework (e.g. the human CDCP1 binding molecule may have a portion of a framework that only contains one or more of the four subregions).

[0024] As used herein, the term "fully human framework" means a framework with an amino acid sequence found naturally in humans. Examples of fully human frameworks, include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA; and Wu et al., (1970) J. Exp. Med. 132, 211-250, both of which are herein incorporated by reference). In certain embodiments, the human CDCP1 binding molecules herein have a fully human framework.Attorney Docket Number: CCF-44473.601

[0025] As used herein, the terms "subject" and "patient" refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human.

[0026] As used herein, the term "codon" or "triplet" refers to a group of three adjacent nucleotides which specify one of the naturally occurring amino acids found in polypeptides. The term also includes codons which do not specify any amino acid. It is also noted that, due to the degeneracy of the genetic code, there are many codons that code for the same amino acid. As such, many of the bases of the nucleic acid sequences of the present invention can be changed without changing the actual amino acid sequence that is encoded. The present disclosure is intended to encompass all such nucleic acid sequences.

[0027] As used herein, the terms "an oligonucleotide having a nucleotide sequence encoding a polypeptide," "polynucleotide having a nucleotide sequence encoding a polypeptide," and "nucleic acid sequence encoding a peptide" means a nucleic acid sequence comprising the coding region of a particular polypeptide. The coding region may be, for example, present in a cDNA, genomic DNA, or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double- stranded. Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript.

[0028] Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.

[0029] Also, as used herein, there is no size limit or size distinction between the terms "oligonucleotide" and "polynucleotide." Both temrs simply refer to molecules composed of nucleotides. Likewise, there is no size distinction between the terms "peptide" and "polypeptide." Both terms simply refer to molecules composed of amino acid residues.

[0030] As used herein, the term "the complement of" a given sequence is used in reference to the sequence that is completely complementary to the sequence over its entire length. For example, the sequence 5'-A-G-T-A-3' is "the complement" of the sequence 3 -T-C-A-T-5'.

[0031] The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide" or "isolated nucleic acid sequence encoding a CDCP1 binding molecule" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated (e.g. host cell proteins).Attorney Docket Number: CCF-44473.601

[0032] As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, CDCP1 binding molecules (e.g., antibodies or antibody fragments) may be purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulins that do not bind to the same antigen. The removal of non-immunoglobulin proteins and / or the removal of immunoglobulins that do not bind the particular antigen results in an increase in the percentage of antigen specific immunoglobulins in the sample. In another example, recombinant antigen-specific polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percentage of recombinant antigen- specific polypeptides is thereby increased in the sample.

[0033] As used herein, the temr "Fc region" refers to a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region (e.g., with increased or decreased effector functions).

[0034] DESCRIPTION OF THE INVENTION

[0035] Provided herein are human CDCP1 binding molecules and nucleic acid sequences encoding such molecules. In particular embodiments, provided herein are human CDCP1 binding molecules (e.g., monoclonal antibodies or antigen binding fragments thereof) having particular light and / or heavy chains variable regions, or light chain and / or heavy chain CDRs, and methods for using such molecules to treat cancer (e.g., colon, bladder, and breast cancer). In some embodiments, the CDCP1 binding molecules are conjugated to a cytotoxin (e.g., via a cleavable linker) so as to allow the specific targeting and delivery of a cytotoxic payload to selectively kill cancerous cells.

[0036] CDCP1 is a type-1 transmembrane protein that is highly expressed on a wide variety of solid tumor cells including those found in triple negative breast cancer, colon cancer, and bladder cancer. Additionally, the expression of this transmembrane protein has been strongly associated with patient prognosis and has demonstrated a critical role in cancer cell survival and metastasis. These observations suggest that CDCP1 may be an effective therapeutic target in these types of cancers. While the mAb-mediated cytotoxic killing of cancer cells has proven to be an effective therapeutic strategy, many tumor cell types have exhibited potent resistance to this mechanism and require the use of an antibody-drug conjugate (ADC). This strategy enables the specific targeting and delivery of a cytotoxic payload to selectively kill cancerous solid tumor cells.Attorney Docket Number: CCF-44473.601

[0037] At present, there exists a severe unmet need in available therapeutic strategies, specifically pertaining to ADC-enabled drug candidates, for CDCP1 expressing cancers including bladder cancer, triple negative breast cancer, and colon cancer. Work conducted during the development of embodiments of the present disclosure have developed an anti-CDCP1 mAh ADC as a treatment for a variety of cancer types including bladder, colon, and triple negative breast cancers. This ADC was developed using a clinically proven mitotic toxin MMAE (Monomethyl auristatin E ). This exemplary antibody and drug conjugate is connected using an acid cleavable linker, allowing it to block its cytotoxic activity until it is internalized by the target tumor cells. The exemplary anti-CDCPl ADC successfully completed in vitro efficacy studies in which it demonstrated potent cancer cell killing activity in triple negative breast cancer and colon cancer cell lines.

[0038] In certain embodiments, the human CDCP1 binding molecules comprise one or more of the antibodies, variable regions, or CDRs shown in SEQ ID NOS:4-6 and 8-10 and / or variable regions or CDRs with one or more conservative or non-conscrvativc amino acid changes in these SEQ ID NOS: 4-6 and 8-10, and nucleic acid sequences encoding SEQ ID NOs: 4-8 and 8-10 (e.g., using at least a portion of the nucleic acid sequences in SEQ ID NOs: 3 or 7). Changes to the amino acid sequences of the CDRs or variable regions (see Figures 5-6) may be generated by changing the nucleic acid sequence encoding the amino acid sequence. A nucleic acid sequence encoding a variant of a given CDR or variable region may be prepared by methods known in the art using the guidance of the present specification for particular sequences. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared nucleic acid encoding the CDR or variable region.

[0039] Briefly, in carrying out site-directed mutagenesis of DNA, the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated in the resulting double-stranded DNA.

[0040] PCR mutagenesis is also suitable for making amino acid sequence variants of the starting CDR (see, e.g., Vallette et. al., (1989) Nucleic Acids Res. 17: 723-733, hereby incorporated by reference). Briefly, when small amounts of template DNA are used as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specificAttorney Docket Number: CCF-44473.601

[0041] DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.

[0042] Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., (1985) Gene 34: 315-323, hereby incorporated by reference. The starting material is the plasmid (or other vector) comprising the starting CDR or variant region DNA to be mutated. The codon(s) in the starting DNA to be mutated are identified. There should be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the starting polypeptide DNA. The plasmid DNA is cut at these sites to linearize it. A double- stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques. This double- stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated DNA sequence.

[0043] Alternatively, or additionally, the desired amino acid sequence encoding a CDR variant, or variable region variant, can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically. Conservative modifications in the amino acid sequences of the CDRs or variable region may also be made. Naturally occurring residues are divided into classes based on common side-chain properties:

[0044] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0045] (2) neutral hydrophilic: cys, ser, thr;

[0046] (3) acidic: asp, glu;

[0047] (4) basic: asn, gin, his, lys, arg;

[0048] (5) residues that influence chain orientation: gly, pro; and

[0049] (6) aromatic: trp, tyr, phe.

[0050] Conservative substitutions will entail exchanging a member of one of these classes for another member of the same class in a particular antibody, variable region, or CDR, such as in SEQ ID NOS: 3-10.

[0051] The CDRs of the present invention may be employed with any type of suitable framework. In some embodiments, the CDRs are used with fully human frameworks, or framework sub-regions. For example, the NCBI web site contains the sequences for knownAttorney Docket Number: CCF-44473.601

[0052] human framework regions. Examples of human VH sequences include, but are not limited to, VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81, which are provided in Matsuda et al., (1998) J. Exp. Med. 188:1973-1975, that includes the complete nucleotide sequence of the human immunoglobulin chain variable region locus, herein incorporated by reference. Examples of human VK sequences include, but are not limited to, Al, A10, Al 1, A14, A17, A18, A19, A2. A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, LI, LIO, LI 1, L12, L14, L15, LI 6, L18, L19, L2, L20, L22, L23, L24, L25, L4 / 18a, L5, L6, L8, L9, 01, OIL 012, 014, 018, 02, 04, and 08, which are provided in Kawasaki et al., (2001) Eur. J. Immunol. 31:1017-1028; Schable and Zachau, (1993) Biol. Chem. Hoppe Seyler 374:1001-1022; and Brensing-Kuppcrs et al., (1997) Gene 191:173-181, all of which arc herein incorporated by reference. Examples of human VL sequences include, but are not limited to, V1-11, V1-13, V1-16, VI-17, Vl-18, Vl-19, Vl-2, Vl-20, Vl-22, Vl-3, Vl-4, Vl-5, Vl-7, Vl-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8, V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, and V5-6, which are provided in Kawasaki et al., (1997) Genome Res. 7:250-261, herein incorporated by reference. Fully human frameworks can be selected from any of these functional germline genes. Generally, these frameworks differ from each other by a limited number of amino acid changes. These frameworks may be used with the CDRs described herein. Additional examples of human frameworks which may be used with the CDRs of the present invention include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA; and Wu et al., (1970), J. Exp. Med. 132:211-250, both of which are herein incoiporated by reference).

[0053] In certain embodiments, the human CDCP1 binding molecules of the present invention comprise antibodies or antibody fragments (e.g., comprising one or more of the CDRs described herein, such as in Figure 6). An antibody, or antibody fragment, of the present invention can be prepared, for example, by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For example, to express an antibody recombinantly, a host cell may be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and.Attorney Docket Number: CCF-44473.601

[0054] preferably, secreted into the medium in which the host cell is cultured, from which medium the antibody can be recovered. Standard recombinant DNA methodologies may be used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al., all of which are herein incorporated by reference.

[0055] In certain antibodies, the anti-CDCPl antibodies, or fragments, thereof prepared herein have an IgG isotype constant regions as shown in Table 1 below.

[0056] TABLE 1

[0057]

[0058] To express an antibody with one or more of the CDRs herein, DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of germline light and heavy chain variable sequences using the polymerase chain reaction (PCR).

[0059] Once the germline VII and VL fragments are obtained, these sequences can be mutated to encode one or more of the CDR amino acid sequences disclosed herein (see, Figure 6). The amino acid sequences encoded by the germline VH and VL DNA sequences may be compared to the CDRs sequence(s) desired to identify amino acid residues that differ from the germline sequences. Then the appropriate nucleotides of the germline DNAAttorney Docket Number: CCF-44473.601

[0060] sequences are mutated such that the mutated germline sequence encodes the selected CDRs, using the genetic code to determine which nucleotide changes should be made. Mutagenesis of the germline sequences may be carried out by standard methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis. In other embodiments, the variable region is synthesized de novo (e.g., using a nucleic acid synthesizer).

[0061] Once DNA fragments encoding the desired VH and VL segments are obtained (e.g., by amplification and mutagenesis of germline VH and VL genes, or synthetic synthesis, as described above), these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another polypeptide, such as an antibody constant region or a flexible linker. The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3). The sequences of mouse and human heavy chain constant region genes are known in the art and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be, for example, an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CHI constant region.

[0062] The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of mouse and human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al., (1991) Sequences of Proteins of immunological Interest, Fifth Edition, U.S.

[0063] Department of Health and Human Services. NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0064] To create a scFv gene, the VH- and VL-encoding DNA fragments may be operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous singlechain protein, with the VL and VH regions joined by the flexible linker (see e.g., Huston etAttorney Docket Number: CCF-44473.601

[0065] al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and McCafferty et al., (1990) Nature 348:552-554), all of which are herein incorporated by reference).

[0066] To express the antibodies, or antibody fragments of the invention, DNAs encoding partial or full-length light and heavy chains, (e.g. obtained as described above), may be inserted into expression vectors such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are generally chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the light or heavy chain sequences, the expression vector may already carry antibody constant region sequences. For example, one approach to converting the VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment(s) within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in- frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a nonimmunoglobulin protein).

[0067] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), herein incorporated by reference. It will be appreciated by those skilled in the art that the design of the expression vector, including theAttorney Docket Number: CCF-44473.601

[0068] selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. In certain embodiments, regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Vims 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma vims. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No.

[0069] 4,510,245 by Bell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al., all of which are herein incorporated by reference.

[0070] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634.665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection / amplification) and the neomycin gene (for G418 selection).

[0071] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains may be transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.

[0072] In certain embodiments, the expression vector used to express the human CDCP1 binding molecules of the present invention are viral vectors, such as retro- viral vectors. Such viral vectors may be employed to generate stably transduced cell lines (e.g. for a continues source of the CDCP1 binding molecules). In some embodiments, the GPEX gene product expression technology (from Catalent, Somerset, NJ) is employed to generate CDCP1 binding molecules (and stable cell lines expressing the CDCP1 binding molecules). In particular embodiments, the expression technology described in W00202783 andAttorney Docket Number: CCF-44473.601

[0073] W00202738 to Bieck et al. (both of which are herein incorporated by reference in their entireties) is employed.

[0074] Mammalian host cells for expressing the recombinant antibodies of the invention include, for example, Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells. In other embodiments, the host cells express GnT III as described in WO9954342 and U.S. Pat. Pub. 20030003097, both herein incoiporated by reference, such that expressed CDCP1 binding molecules have increased ADCC activity. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are generally produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells arc grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0075] Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain of an antibody of this disclosure. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to CDCP1. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bi-functional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than CDCP1 (e.g., by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods).

[0076] In certain embodiments, the human CDCP1 binding molecules of the present invention (e.g., as antibodies or antibody fragments) are useful for immunoassays which detect human CDCP1 expressing cells in a sample (e.g., a purified blood sample from a subject). In some embodiments, an immunoassay CDCP1 typically comprises incubating a biological sample in the presence of a detectably labeled antibody or antibody fragment of the present invention capable of selectively binding to CDCP1 present on cells, and detecting the labeled peptide or antibody which is bound in a sample. Various clinical assay proceduresAttorney Docket Number: CCF-44473.601

[0077] are well known in the art. Any suitable assay known in the art can be used in such a method. Examples of such assays include, but are not limited to, immunoassay, such as sandwich immunoassay (e.g., monoclonal-polyclonal sandwich immunoassays, including radioisotope detection (radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.)), competitive inhibition immunoassay (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT), an ARCHITECT assay (ABBOTT), a bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescent assay, etc.

[0078] A human CDCP1 binding molecule can be captured on beads or nitrocellulose, or on any other solid support which is capable of immobilizing soluble proteins (e.g., magnetic beads). A human CDCP1 containing sample (e.g., cells expressing CDCP1 on their surface) is then added to the support which is subsequently washed with suitable buffers to remove unbound proteins. A second, delectably labeled, molecule (e.g., antibody or peptide) that can bind to CDCP1 binding molecule is added to the solid phase support that can then be washed with the buffer a second time to remove unbound molecules. The amount of bound label on the solid support can then be detected by known methods.

[0079] Detectably labeling the CDCP1 binding molecule can be accomplished by coupling to an enzyme for use in an enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts with the exposed substrate to generate a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the human CDCP1 binding molecules of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta- 5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0080] In some embodiments of the present invention, CDCP1 which is detected by the above assays can be present in a biological sample (e.g., containing cancer cells, such as colon, bladder, or breast cancer cells). Any sample containing human CDCP1 can be used. In certain embodiments, the sample is a biological fluid such as, for example, blood, brain tissue, serum, lymph, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, a tissue extract or homogenate, and the like. However, the invention is not limited to assays using only theseAttorney Docket Number: CCF-44473.601

[0081] samples, as it is possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.

[0082] In certain embodiments, provided here are kits for the detection of CDCP 1 that include a human CDCP1 detection molecule. Such kits may include any of the immunodiagnostic reagents described herein and may further include instructions for the use of the immunodiagnoslic reagents in immunoassays for determining the presence of human CDCP1 in a test sample (e.g., a test sample containing CDCP1 expressing cancer cells). The kits may also include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and / or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.

[0083] The various components of the kit may be provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instrument for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.

[0084] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, medicine, and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

Attorney Docket Number: CCF-44473.601CLAIMS:We claim:

1. A composition comprising a human CDCP1 binding molecule, and / or one or more nucleic acid molecules encoding said human CDCP1 binding molecule,wherein said human CDCP1 binding molecule comprises:a) a heavy chain variable region, wherein said heavy chain variable region comprises:i) a CDRH1 amino acid sequence comprising SEQ ID NO: 4, or SEQ ID NO:4 with one with one or two conservative amino acid changes,ii) a CDRH2 amino acid sequence comprising SEQ ID NO: 5, or SEQ ID NO: 5 with one or two conservative amino acid changes, andiii) a CDRH3 amino acid sequence comprising SEQ ID NO: 6, or SEQ ID NO: 6 with one with one or two conservative amino acid changes, and / orb) a light chain variable region, wherein said light chain variable region comprises;i) a CDRL1 amino acid sequence comprising SEQ ID NO: 8, or SEQ ID NO: 8 with one with one or two conservative amino acid changes,ii) a CDRL2 amino acid sequence comprising SEQ ID NO: 9, or SEQ ID NO: 9 with one with one or two conservative amino acid changes, andiii) a CDRL3 amino acid sequence comprising SEQ ID NO: 10, or SEQ ID NO: 10 with one with one or two conservative amino acid changes; and optionally, wherein said human CDCP1 binding molecule is linked to a cytotoxic agent via a linker, and optionally wherein said linker is cleaved when it is internalized in a cell.

2. The composition of claim 1, wherein:i) said CDRH1 amino acid sequence comprises SEQ ID NO: 4;ii) said CDRH2 amino acid sequence comprises SEQ ID NO: 5;iii) said CDRH3 amino acid sequence comprises SEQ ID NO: 6;iv) said CDRL1 amino acid sequence comprises SEQ ID NO: 8;v) said CDRL2 amino acid sequence comprises SEQ ID NO: 9; andvi) said CDRL3 amino acid sequence comprises SEQ ID NO: 10.Attorney Docket Number: CCF-44473.6013. The composition of Claim 1, wherein said human CDCP1 binding molecule is an antibody, minibody, diabody, scFv, or antibody fragment capable of binding human CDCP1.

4. The composition of Claim 3, wherein said antibody fragment is a Fab, F(ab')2 or Fv antibody fragment.

5. The composition of Claim 2, wherein said antibody or antibody fragment comprises at least an antigen binding portion of the 9A2 antibody6. The composition of Claim 1, wherein said heavy chain and / or light chain variable region comprises a human framework region.

7. The composition of Claim 1, wherein said human CDCP1 binding molecule further comprises a light chain constant region and a CHI heavy chain constant region.

8. The composition of Claim 7, wherein said CDCP1 binding molecule further comprises a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

9. The composition of Claim 8, wherein said light chain constant region is human or a humanized murine, and / or wherein said CHI, CH2, and CH3 heavy chain constant regions are human or are humanized murine.

10. The composition of Claim 1, wherein said human CDCP1 binding molecule comprises an antibody, wherein the light chain constant region of said antibody is selected from: IgG Kappa and IgG Lambda, and wherein the heavy chain constant region of said antibody is selected from: IgGl, IgG2, IgG3, and IgG4.

11. The composition of Claim 1, wherein said human CDCP1 binding molecule comprises an antibody, or antigen binding portion thereof, which is glycosylated or nonglycosylated.

12. The composition of Claim 1, further comprising a physiologically tolerable buffer.Attorney Docket Number: CCF-44473.60113. The composition of Claim 1, wherein said heavy chain variable regions comprises SEQ ID NO: 3, or SEQ ID NO:3 with one or more conservative amino acid changes.

14. The composition of Claim 1, wherein said light chain variable region comprises SEQ ID NO: 7, or SEQ ID NO:7 with one or more conservative amino acid changes.

15. The composition of Claim 1, wherein said composition comprises said one or more nucleic acid molecules.

16. The composition of Claim 1, wherein said one or more nucleic acid molecules comprise: i) a first nucleic acid sequence encoding said heavy chain variable region, and ii) a second nucleic acid sequence encoding said light chain variable region.

17. The composition of Claim 16, further comprising an expression vector, and wherein said first and / or second nucleic acid sequences are present in said expression vector.

18. A method of treating or preventing cancer in a subject with cancer comprising: treating a subject with a human CDCP1 binding molecule, or one or more mRNAs encoding said CDCP1 binding molecules, or an expression vector comprising one or more nucleic acid molecules encoding said human CDCP1 binding molecule, optionally wherein said human CDCP1 binding molecule is as recited in any of Claims 1-17.

19. The method of Claim 18, wherein said subject has colon, bladder or breast cancer, and optionally wherein said breast cancer is triple negative breast cancer.

20. The method of Claim 18, wherein said human CDCP1 binding molecule is an antibody or antigen binding portion thereof.

21. The method of Claim 18, wherein said antibody or antigen binding portion thereof is a human antibody or antigen binding portion thereof.

22. The method of Claim 18, wherein said antibody or antigen binding portion thereof is a humanized antibody or antigen binding portion thereof.Attorney Docket Number: CCF-44473.60123. The method of Claim 18, wherein said heavy chain variable region of said human CDCP1 binding molecule comprises SEQ ID NO: 3, or SEQ ID NO:3 with one or more conservative amino acid changes.

24. The composition of Claim 18, wherein said light chain variable region of said human CDCP1 binding molecule comprises SEQ ID NO: 7, or SEQ ID NO: 7 with one or more conservative amino acid changes.

26. A method of detecting CDCP1 -positive cells in a sample comprising:a) contacting a sample with anti-CDCPl antibodies, or antigen binding portions thereof,wherein said anti-CDCPl antibodies, or antigen binding portions thereof, form a complex with CDCP1 -positive cells if present in said sample; andb) detecting the presence or absence of said complex in said sample.

27. The method of Claim 26, wherein said sample is from a subject that has, or is suspected to develop, cancer.

28. The method of Claim 26, wherein said human anti-CDCPl antibodies, or antigen binding portions thereof, comprise a detectable label, and / or wherein said anti-CDCPl antibodies, or antigen binding portions thereof, are as recited in Claim 1-17.

29. The method of Claim 26, further comprising contacting said sample with a conjugate molecule capable of binding to said anti-PCA3 antibodies, or antigen binding portions thereof, wherein said conjugate molecule comprises a detectable label.

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