Compositions related to gender selection and methods of use

Abstract

Aspects of this invention relate to a novel monoclonal antibody specifically designed to exhibit selective binding affinity towards antigens present predominantly in X chromosome sperm. The antibody is engineered for applications to sex select sperm for cattle breeding purposes.

Description

COMPOSITIONS RELATED TO GENDER SELECTION AND METHODS OF USE PRIORITY PARAGRAPH

[0001] This Application claims priority to US Provisional Application 63 / 739,666 filed 12 / 29 / 2024 which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] None.REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing is being submitted electronically with this application. The sequence listing is incorporated herein by reference. The sequence listing that is contained in the file named "BIOSPOOOIWO" which is 137,678 bytes (as measured in Microsoft Windows®) and was created on 12 / 29 / 2025.FIELD

[0004] The invention is related to the field of agriculture and animal husbandry, specifically monoclonal antibodies for sexed semen production.BACKGROUND

[0005] Gender selection in cattle, often referred to as sexed semen technology, has become increasingly important in modern dairy and beef cattle farming for several reasons. Traditionally, farmers relied on natural mating or artificial insemination (Al) with no control over the sex of the offspring. This led to a roughly 50:50 split between male and female calves. In dairy farming, females are highly desirable because they produce milk, whereas males, unless destined for beef or breeding, are often considered less valuable. In beef farming, the preference might lean towards males for meat production, but the need for replacement heifers is also crucial. Sexed semen technology was initiated in the early 2000s where X (female) sperm were separated from Y (male) sperm based on the fact that X sperm are slightly heavier. Current sex sorting methods include (1) flow cytometry where sperm cells are stained with a DNA-specific dye, the DNA in X sperm absorbs more dye than Y sperm, allowing for sorting based on fluorescence intensity;and (2) microfluidic sorting where sperm flow through microchannels where X and Y sperm are separated by their size and shape.

[0006] U.S. Patent 11,760,793 entitled “Antibody for skewing sex ratio and methods of use thereof’ issued September 19, 2023 describes rabbit polyclonal antibodies generated against GX1-E or GX1-M protein. Polyclonal serum, while useful in various applications, especially in research and diagnostics, has several disadvantages such as (1) batch-to-batch variability due to production by multiple B cell clones leading to variability in the composition of antibodies between different batches; (2) cross-reactivity, due to the nature of polyclonal antibodies there is a higher risk of cross-reactivity with unintended antigens leading to false positives / binding where high specificity is crucial; (3) limited supply, since polyclonal antibodies are often derived from immunized animals (like rabbits, goats, or sheep) once the animal is no longer available (due to death or end of production cycle) the exact antibody mix cannot be replicated limiting long-term availability for ongoing or future studies; (4) purity, purifying polyclonal antibodies to remove non-specific or unwanted antibodies can be complex and less effective than with monoclonal antibodies potentially leading to higher background noise in applications; (5) antigenic competition, in a mixture of polyclonal antibodies some antibodies might compete with others for binding sites on the antigen possibly reducing the overall effectiveness of the serum in binding to a target; and (6) risk of contamination, there is a contamination risk with animal pathogens or other proteins from the serum or plasma which might require additional purification steps or testing to ensure safety for use. Furthermore, when dealing with polyclonal antibodies there is less control over which epitopes on the antigen are recognized which can be a disadvantage in scenarios where targeting a specific epitope is necessary for function.

[0007] While sexed semen is more expensive than conventional semen, the ability to produce more of the desired sex can lead to cost savings in the long run by reducing the number of unwanted calves. Also, sexed semen allows for more strategic use of high-genetic merit males, accelerating genetic progress in herds.

[0008] Sexed semen technology in cattle and other species has fundamentally changed how farmers can manage their herds or livestock, offering tools to align production with economic needs more precisely. There is a need for additional methods and compositions for use in sexed semen technology and application thereof.SUMMARY

[0009] To address some of the problems alluded to above, this application describes the development of monoclonal antibodies for use in producing sexed semen for use in cattle reproduction. The invention includes, in certain embodiments, anti-GXl antibodies, or antigen binding portions thereof.

[0010] Certain embodiments are directed to a monoclonal antibody or antibody fragment that selectively binds to GX1, said antibody comprising: (i) heavy chain CDRs as set forth in SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69 and light chain CDRs as set forth in SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; (ii) heavy chain CDRs as set forth in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 and light chain CDRs as set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58; (iii) heavy chain CDRs as set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 and light chain CDRs as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18; (iv) heavy chain CDRs as set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109 and light chain CDRs as set forth in SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118; (v) heavy chain CDRs as set forth in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 and light chain CDRs as set forth in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; or (vi) heavy chain CDRs as set forth in SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89 and light chain CDRs as set forth in SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98.

[0011] In certain aspects the monoclonal antibody or antibody fragment, wherein an antibody or antibody fragment comprises (i) a heavy chain variable region that is at least 90% identical to SEQ ID NO:66 and light chain variable region that is at least 90% identical to SEQ ID NO:75; (ii) a heavy chain variable region that is at least 90% identical SEQ ID NO:46 and light chain variable region that is at least 90% identical SEQ ID NO:55; (iii) a heavy chain variable region that is at least 90% identical SEQ ID NO:6 and light chain variable region that is at least 90% identical SEQ ID NO: 15; (iv) heavy chain variable region that is at least 90% identical SEQ ID NO: 106 and light chain variable region that is at least 90% identical SEQ ID NO: 115; (v) a heavy chain variable region that is at least 90% identical SEQ ID NO:26 and light chain variable region that is at least 90% identical SEQ ID NO:35; or (vi) a heavy chain variable region that is at least 90% identical SEQ ID NO:86 and light chain variable region that is at least 90% identical SEQ ID NO:95. A monoclonal antibody can comprise 1, 2, 3, or more amino acid substitutions in one or more CDRs as long as the binding specificity remains. The monoclonal antibody orantibody fragment can include a heterologous moiety forming an antibody or antibody fragment conjugate. In certain aspects the antibody or antibody fragments can be coupled to a particle, such as a polymeric particle, magnetic particle or a surface. Certain compositions can include 1, 2, 3, 4, 5, 6 or more separate monoclonal antibodies or fragments thereof that are described herein, i.e., the antibodies can be pooled and used concurrently. Alternatively, the various antibodies or fragments can be used in series in various combinations.

[0012] Certain embodiments are directed to methods of selecting sex specific sperm comprising: (i) contacting a sperm composition with one or more antibody or antibody fragments as described herein forming an X chromosome containing sperm complex and a non-complexed Y chromosome containing sperm; and (ii) isolating the X chromosome containing sperm complex to form an enhanced X chromosome containing sperm composition and / or isolating the non-complexed Y chromosome containing sperm to form an enhanced non-complexed Y chromosome containing sperm composition. The method can include evaluating the enhanced X chromosome containing sperm composition and / or the enhanced non-complexed Y chromosome containing sperm composition for one or more of motility or morphology. The method can also include preserving the enhanced X chromosome containing sperm composition and / or the enhanced non-complexed Y chromosome containing sperm composition. The enhanced X chromosome containing sperm composition and / or the enhanced non-complexed Y chromosome containing sperm composition can be frozen or preserved.

[0013] Other embodiments are directed to methods of inseminating a mammal (e.g., bovine, porcine, etc.) comprising depositing an enhanced X chromosome containing sperm composition or an enhanced Y chromosome containing sperm composition into the reproductive tract of a female (e.g., a uterus of a female) or alternatively into an in vitro container comprising ovum. Mammals can include livestock or wildlife. Animals that may be subjected to artificial insemination as described herein include Cattle (Bovine), Pigs (Swine), Sheep (Ovine), Horses (Equine), Goats (Caprine), Dogs (Canine), Cats (Feline), Camels / Llamas (Cameline), Deer (Cervidine), and endangered species such as Elephant (Elephantidae), Rhinoceros (Rhinocerotidae), and the like. The sperm may also be used in in vitro fertilization (IVF) procedures.

[0014] Certain aspects are directed to methods of fertilizing a mammalian egg / ovum comprising contacting an egg / ovum in vivo, in vitro or ex vivo with an enhanced X chromosomecontaining sperm composition or an enhanced non-complexed Y chromosome containing sperm composition forming a fertilized egg. The method can further comprise implanting the fertilized egg in a recipient female.

[0015] Certain embodiments are directed to isolated nucleic acids encoding antibodies, or antigen binding portions thereof as described herein.

[0016] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0017] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0018] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0019] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0020] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a nonexclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components.

[0021] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used ina claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0022] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0024] Definition of terms used herein include:

[0025] The term “antibody” broadly refers to an immunoglobulin (Ig) molecule, generally comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential target binding features of an Ig molecule. Such fragment, mutant, variant, or derivative antibody formats are known in the art.

[0026] In a full-length antibody, 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 light 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 (CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) and class (e.g., IgGl, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.

[0027] The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The system described by Kabat provides an unambiguous residue numbering system applicable to any variable region of an antibody but also provides precise residue boundaries defining the three CDRs.

[0028] The framework and CDR regions of an antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized by substitution, insertion and / or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. Shorter sequences can have 1, 2, 3, 4, or more amino acid substitutions while maintaining binding specificity.

[0029] “Percent (%) amino acid sequence identity” with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0030] The term “antigen binding portion” of an antibody (or simply “antibody portion”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Such antibody embodiments may also be bispecific, dual specific, or multi-specific formats specifically binding to two or more different antigens. Examples of binding fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHIdomains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). The Fv fragment, VL and VH, 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 regions pair to form monovalent molecules (known as single chain Fv (scFv)). In certain embodiments of the invention, scFv molecules may be incorporated into a fusion protein. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific 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 antigen binding sites. Such antibody binding portions are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer- Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

[0031] The term “antibody construct” as used herein refers to a polypeptide comprising one or more the antigen binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Such linker polypeptides are well known in the art (see e g., Holliger et al. (1993) PNAS USA 90:6444-48; Poljak, et al. (1994) Structure 2:1121-23). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Exemplary human IgG heavy chain and light chain constant domain amino acid sequences are known in the art and represented below.

[0032] The terms “Kabat numbering,” “Kabat definitions,” and “Kabat labeling” are used interchangeably herein. These terms refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0033] The term “multivalent antibody” is used herein to denote an antibody comprising two or more antigen binding sites. In certain embodiments, the multivalent antibody may be engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody.

[0034] The term “epitope” refers to a region of an antigen that is bound by an antibody or antibody fragment or antibody conjugate. In certain embodiments epitopes have specific three-dimensional structural characteristics (conformational epitope). In certain embodiments, an antibody is said to specifically bind an antigen when it can preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0035] The term “labeled antibody” or “antibody conjugate” as used herein, refers to an antibody, or an antigen binding portion thereof, with a label or heterologous moiety incorporated or coupled to an antibody or antibody fragment. The label can be a detectable marker, e.g., incorporation of a fluorophore, a radiolabeled amino acid, or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Antibodies can be conjugated to nanoparticles / particles (metal or polymeric) or surfaces for detection, imaging, biosensor, or purification / isolation applications. Antibodies can be directly conjugated to magnetic beads or polymer beads for cell or sperm isolation methods using gravitational, magnetic-activated or other cell sorting methods. Antibody conjugates leverage the specificity of antibodies to target particular cell or gamete types, combined with the unique properties of the conjugated molecules for detection, separation, or other functional purposes. Each type of conjugate has specific applications based on the nature of the conjugated molecule and the intended use in cell purification or isolation protocols.

[0036] “Functional fragments” or “antibody fragments” of such antibodies comprise portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. Functional fragments include but are limited to antibodies with multiple epitope specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as Fab, F(ab’)2, Fd, Fabc, and Fv molecules, single chain (Sc) antibodies (also called ScFv), individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, dimers consistingof one heavy and one light chain, and the like. The antibodies and functional antibody fragments described herein bind an epitope of GX1 (SEQ ID NO: 121).

[0037] An “anti-GXl” antibody or antibody fragment as described herein specifically binds GX1. An “anti-GXl antibody” includes the mAb216-878A9 (878A9), mAb216-878G9 (878G9), mAb216-878H3 (878H3), mAb216-878H5 (878H5), mAb216-879Fl (879F1), or mAb216-961D12 (961D12), including antibody fragments thereof.

[0038] In certain aspects an antibody can comprise a heavy chain variable region as set forth in SEQ ID NO: 66 and light chain variable region SEQ ID NO: 75. In certain aspects an antibody or antibody fragment can comprise heavy chain CDRs as set forth in SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69 and a light chain CDRs as set forth in SEQ ID NO: 76, SEQ ID NO: 77, and SEQ ID NO: 78.

[0039] In certain aspects an antibody can comprise a heavy chain variable region as set forth in SEQ ID NO: 46 and light chain variable region SEQ ID NO: 55. In certain aspects an antibody or antibody fragment can comprise heavy chain CDRs as set forth in SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49 and a light chain CDRs as set forth in SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO: 58.

[0040] In certain aspects an antibody can comprise a heavy chain variable region as set forth in SEQ ID NO: 6 and light chain variable region SEQ ID NO: 15. In certain aspects an antibody or antibody fragment can comprise heavy chain CDRs as set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 and a light chain CDRs as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0041] In certain aspects an antibody can comprise a heavy chain variable region as set forth in SEQ ID NO: 106 and light chain variable region SEQ ID NO: 115. In certain aspects an antibody or antibody fragment can comprise heavy chain CDRs as set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109 and a light chain CDRs as set forth in SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118.

[0042] In certain aspects an antibody can comprise a heavy chain variable region as set forth in SEQ ID NO: 26 and light chain variable region SEQ ID NO: 35. In certain aspects an antibody or antibody fragment can comprise heavy chain CDRs as set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29 and a light chain CDRs as set forth in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 3.

[0043] In certain aspects an antibody can comprise a heavy chain variable region as set forth in SEQ ID NO: 86 and light chain variable region SEQ ID NO: 95. In certain aspects an antibody or antibody fragment can comprise heavy chain CDRs as set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89 and a light chain CDRs as set forth in SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.DESCRIPTION OF THE DRAWINGS

[0044] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0045] FIG. 1. Gx Sex skewing in Porcine semen from 3 boars. Using the same techniques as explained for bovine semen, Gx 1 positive cells resulted in 74% female (XX) bearing sperm. Sperm following post skewing had normal motility and morphology, similar to what has been seen in bovine cells. These results suggest that Gx is conserved in porcine and has similar features as what is reported in bovine.DESCRIPTION

[0046] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any one embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that the following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0047] The sex of a mammal is determined by a pair of sex chromosomes. Females exhibit two X chromosomes and Males exhibit an X and a Y chromosome. Methods of influencing sex ratio by influencing sperm sex ratio (sex selecting sperm) are desirable. A current method for sex selecting sperm involves the use of flow cytometry, a technique that sorts sperm based on the difference in DNA content between X-chromosome-bearing sperm (which produce femaleoffspring) and Y-chromosome-bearing sperm (which produce male offspring). Sperm cells are stained with a fluorescent dye which binds to DNA. Since X-chromosome-bearing sperm contain about 4% more DNA than Y-chromosome-bearing sperm, they will take up more dye and thus fluoresce brighter when exposed to UV light. The stained sperm are then passed through a flow cytometer where they are aligned in a single stream. Each sperm is encapsulated in a droplet of fluid. As the sperm pass through a laser beam, the amount of fluorescence emitted by each sperm is measured. Based on the fluorescence intensity, the sperm are sorted into higher fluorescence (X-sperm) and lower fluorescence (Y-sperm).

[0048] Embodiments of the current invention are directed antibody based separation methods. Various aspects of the invention relate to anti-GXl monoclonal antibodies (GX1 having an amino acid sequence MTKRTGKPQGRVVRKHLPPVTRDKR MKTSSQLRPPKNVKVARASARVNNHLRAKLTKKTSQKPPTTRNLRKNGGSKLCSQCCK VNEELNQNGPEEVPESVEIPVIPAGPVGSQ (SEQ ID NO: 121)) and antibody fragments, anti-GXl monoclonal antibody conjugates (ADCs), and formulations thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. In certain embodiments, the anti-GXl antibody binding portion is a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, an scFv, a single domain antibody, or a diabody.

[0049] Antibodies may be produced by any of a number of techniques. For example, expression from host cells, wherein expression vector(s) encoding the heavy and light chains is (are) 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. Although it is possible to express antibodies in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferable, and most preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

[0050] Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77:4216-20, used with a DHFR selectable marker, NS0 myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodies are 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 are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0051] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules.I. Sexed Semen Technology

[0052] Challenges in this area include lower conception rates with sexed semen compared to conventional semen due to the sorting process potentially reducing sperm viability. The technology and processing are more expensive, which can be a barrier for smaller farms. While highly effective, no method is 100% accurate, and there is always a possibility that a small percentage of the opposite sex is born. There is ongoing research to increase the accuracy of sex sorting and to maintain or improve conception rates.

[0053] Embodiments of the current invention utilize binding a monoclonal antibody that specifically binds X chromosome sperm or its precursor(s) for either capture and isolation of X chromosome sperm resulting in an enhancement of X chromosome sperm or separation of X chromosome sperm from Y chromosome sperm resulting in an enhancement of Y chromosome sperm. The binding of the monoclonal antibodies may also affect the function of the bound sperm. The monoclonal antibody can be conjugated to a moiety that assists in purification or sex selection including a fluorophore for sorting, an affinity tag for adsorption / desorption, a magnetic / metal tag for magnet assisted sorting, a cytotoxic agent to kill X chromosome fertilized oocytes, a blocking agent to hinder fertilization by X chromosome sperm, and the like. In certain aspects sperm can be isolated using antibody-linked Sepharose or similar beads. Sepharose beads can covalently linked with antibodies specific to certain sperm surface proteins or antigens and used in a column or in a batch system. Bound sperm can be eluted from the beads if desired. In certain aspects superparamagnetic particles can be used for sperm isolation through magnetic-activated cell sorting (MACS). The superparamagnetic particles are coated with antibodies that specifically bind to antigens or receptors on the surface of target cells / gametes. When mixedwith a cell sample, these particles attach only to the sperm displaying the target marker, which are then isolate using the physical properties of the superparamagnetic particles / beads.

[0054] In certain aspects, separation can be achieved via positive selection (isolating GX1-bound X-sperm complexes, e.g., via conjugated particles) or negative selection (depleting X-sperm to enrich unbound Y-sperm). Positive selection typically yields higher-purity X-enriched fractions but may involve greater cell loss (about 18-20%) due to bound sperm entrapment. Negative selection minimizes loss in the Y-enriched fraction but can result in lower purity (about 7-9% cross-contamination). Combinations (e.g., sequential positive / negative steps or pooled antibodies) optimize yield and skew (e.g., >80% desired sex while recovering >80% total motile sperm).

[0055] For artificial insemination (Al), sex-sorted sperm may be used. Typically, about 2 to 4 million sorted sperm per inseminate are used. Artificial insemination (Al) in cattle is a reproductive technology used to improve the genetic quality of herds, increase breeding efficiency, and manage the spread of diseases. General steps for insemination include: Collecting semen from males (e.g., bulls), often those with desirable traits (e.g., milk production, meat quality, fertility, or other characteristics). Semen collection can occur via an artificial vagina, electroejaculation, or other methods. Once collected, the semen is evaluated for quality (motility, concentration, morphology of sperm). The collected semen can be subjected to sex selection procedures using one or more antibodies described herein. The semen can be diluted with extenders to increase volume, preserve viability, and allow for multiple inseminations from one collection. The processed semen is then packaged into straws, typically containing about 0.5 ml of extended semen, and frozen in liquid nitrogen at -196°C for storage.

[0056] Females (e.g., Cows) are observe for signs of estrus (heat), which include behavioral changes like standing to be mounted, increased activity, or vocalization. Al is most effective if performed 12 to 24 hours after the onset of estrus, with optimal timing being 12-18 hours post-estrus detection for the highest conception rates. Once respective cows are identified a semen straw is removed from liquid nitrogen storage and thawed at controlled temperatures, usually at body temperature (37°C) for about 30 to 45 seconds. An insemination gun, lubricant, and disposable gloves are prepared. The female is positioned so that her reproductive tract is accessible; this might involve restraining her in a headlock or chute. In certain methods, the inseminator inserts the gloved, lubricated hand into the rectum to manipulate the reproductiveorgans. The insemination gun, loaded with the straw, is then gently passed through the vulva into the vagina. Using the hand in the rectum, the technician guides the gun through the cervix into the uterus, aiming to deposit the semen into the uterine body or horns, depending on the technique preferred or the female’s (e.g., cow's) reproductive anatomy. After insemination, records are kept of which female (e.g., cow) was inseminated, the semen used, and the date. The inseminated females are observed for signs of pregnancy or the return to estrus.

[0057] In Vitro Fertilization (IVF) is a form of assisted reproductive technology (ART) where an egg is fertilized by sperm outside the body. In general the process includes one or more of: (1) ovarian stimulation, for example administering hormone medications to stimulate the ovaries to produce multiple eggs rather than just one; (2) egg retrieval, once the eggs are mature, they are retrieved from the ovaries; (3) sperm collection / preparation (the collected sperm can be subjected to sex selection procedures using one or more antibody described herein); (4) Fertilization, the eggs and sperm are combined; (5) embryo culture, after fertilization, the embryos are cultured for several days (usually 3-5 days); (6) embryo selection and transfer, one or more embryos are selected based on their quality and development stage and the selected embryos are then transferred into the uterus; (7) luteal phase support, post-transfer, medications like progesterone are often given to support the uterine lining and help maintain a pregnancy until the placenta can take over. After the embryo transfer, a pregnancy test is performed to check if the process was successful. Any viable, unused embryos might be cryopreserved (frozen) for future use.

[0058] In vitro experiments in bovine semen can demonstrated effective separation using antibodies conjugated to non-magnetic supports, such as mesh fdters or porous styrofoam / polymeric beads. Sperm suspensions are incubated with antibody-conjugated beads (e.g., styrofoam beads of 50-500 pm diameter), allowing GXl-positive X-sperm to bind. The mixture is then passed through a mesh (e.g., 20-100 pm pore size) or column, where bound X-sperm are retained, and unbound Y-sperm flow through. Retained X-sperm can be eluted by gentle agitation or pH / competition buffers. This low-shear method preserves motility and morphology better than magnetic pulling in some cases.IT. Monoclonal Antibodies and Antibody Fragments that bind GX1

[0059] GX1 has amino acids as set forth in SEQ ID NO: 121. Monoclonal antibodies and / or antibody fragments of this invention are prepared using, for example, techniques that provide for the production of antibody molecules by continuous cell lines in culture including, but not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al. 1975, Nature 256:495-97; Kozboretal, 1985, J. Immunol). Hybridoma sequencing was performed using an Illumina next generation sequencing (NGS) based technology. RNA samples were used to generate a cDNA library that were sequenced, e.g., on an Illumina Novaseq platform. De novo assembly was used to reconstruct the sample transcriptome from raw data. Variable domain sequences were identified by comparison with known sequences. Leader and variable regions were assessed for the quality and completeness of their corresponding sequences.

[0060] In certain aspects, protein structure prediction software was used to identify potential recombinant protein for use as an antigen. GX1 alone or a recombinant fusion protein were considered. The antigen was produced in mammalian cells expressing a His-GST-GXl-HA protein. The protein was purified and used to immunize animals.

[0061] Binding of full monoclonal antibodies may cause steric hindrance, potentially reducing motility of bound X-sperm by physically impeding flagellar movement. This can be mitigated by using antibody fragments (e.g., Fab, scFv), lower antibody densities on particles, or optimized incubation conditions. In porcine studies, no significant motility reduction was observed post-separation, indicating hindrance is manageable.A. mAb216-878H3

[0062] Monoclonal antibody mAb216-878H3 having heavy chain and light sequences of Ighg2a_i9464 CTAGATGTGTTTCCTGTGATTTCTAAAGTCTTATTGCTCTCTTATTGGA GACTCACACTATAGGAAGCCAGAGACCATGATGGTCTTACTTTAATAACCAAGGGC ATTCATTATTTACCTTCCCCAATTATGAAGGCTGGGCTGTCTCCCTGCATGCAAATGC TTCTTCTAACTCTAAGTTAAATCCCCTCTTGGGGTGTGAAAGCTCACATCTCTCTCAT TAGAGGTTGATCTTTGAGGAAAACAGGGTGTTGCCTAAAGGATGAAAGTGTTGAGT CTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTGATGTACAGCTTCAGGAGT CAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAA ACTGGAATGGATGGGCTACATAAGCTACGATGGTAGCAATAACTACAACCCATCTCT CAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTT GAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGGGGGGTACTGG GGGATATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACA ACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAAGTGGCTCCTCG GTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGG AACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGAC CTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCC ATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGA GCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCT CTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGAT CTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAG ATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAA ACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAG CACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCT CCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCAC AGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTG ACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAAC GGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTC TTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATA GCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCT TCTCCCGGACTCCGGGTAAATGAGCTCAGCACCCACAAAACTCTCAGGTCCAAAGA GACACCCACACTCATCTCCATGCTTCCCTTGTATAAATAAAGCACCCAGCAATGCCT GGGACCATGTAAAAAAAAAAAAAAAATAAAAAAGGACAAAAAATTACCACAAGAA AACAAACCCCCAAATATGATAACAAAAAACA (SEQ ID NO: 1) and Igk_i2233 TGAAGTCAAGACTCAGCCTGGACATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTT GCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACAGACTTCATCCTCCCT GTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTA GCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCT ACCACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGATCAAGAAGATATTGCCACTTACT TTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA ATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAG TTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGAC ATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAG TTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGT TGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAG ACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGAGACAAAG GTCCTGAGACGCCACCACCAGCTCCCCAGCTCCATCCTATCTTCCCTTCTAAGGTCTT GGAGGCTTCCCCACAAGCGACCTACCACTGTTGCGGTGCTCCAAACCTCCTCCCCAC CTCCTTCTCCTCCTCCTCCCTTTCCTTGGCTTTTATCATGCTAATATTTGCAGAAAATA TTCAATAAAGTGAGTCTTTGCACTTGAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 2) was developed.

[0063] A polynucleotide encoding the mAb216-878H3 heavy chain includes the nucleic acid sequence includes a (1) heavy chain variable domain signal peptide encoding DNA sequence ATGAAAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCT (SEQ ID NO:3) that encodes a heavy chain variable domain signal peptide protein sequence MKVLSLLYLLTAIPGILS (SEQ ID NO: 4); (2) The heavy chain variable domain encoding nucleic acid sequence is GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAA CCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTT ATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTAC ATAAGCTACGATGGTAGCAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATC ACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAG GACACAGCCACATATTACTGTGCAAGGGGGGTACTGGGGGATATGGACTACTGGGG TCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCC ACTGGCCCCTGTGTGTGGAGATACAAGTGGCTCCTCGGTGACTCTAGGATGCCTGGT CAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAG TGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTC AGTGACTGTAACCTCGAGC (SEQ ID NO: 5) that encodes the heavy chain variable domain having an amino acid sequence of DVQLQESGPGLVKPSOSLSLTCSVTGYSITSGYYW N1WIROFPGNKI.F.WMGYISYDGSNNYNPS[.KN2RISITRDTSI<NOFFI.I<[.NSVTTF.DTATYYCARGVLGDMDY3WGOGTSVTVSSAKTTAPSVYPLAPVCGDTSGSSVTLGCLVKGYF PEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSS (SEQ ID NO: 6) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 7), 2=CDR2 (SEQ ID NO: 8), 3=CDR3 (SEQ ID NO: 9); (3) the heavy chain constant domain ACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCC CACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAA TCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCG TCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGT CACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGT TTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTAC AACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGT GGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAG AACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCC ACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACT TCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAAC TACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAG CTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGT CCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTA AATGA (SEQ ID NO: 10) encoding heavy chain constant domain protein sequence TWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLM ISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQD WMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVT DFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSV VHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 11).

[0064] A polynucleotide encoding the mAb216-878H3 has a (1) light chain variable domain signal peptide DNA sequence ATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTT TTCAAGGTACCAGATGT (SEQ ID NO: 12) encoding light chain variable domain peptide protein sequence MSSAQFLGLLLLCFQGTRC (SEQ ID NO: 13); (2) Light chain variable domain DNA sequence GATATCCAGATGACACAGACTTCATCCTCCCTGTCTGCCTCT CTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTT AAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCACACATC AAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGATCAAGAAGATATTGCCACTTACTTTTGCCAAC AGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA(SEQ ID NO: 14) encoding light chain variable domain sequence DIOMTOTSSSLSASLGDRVTISCRASODISNYLN' WYOOKPDGTVI<LLIYHTSRLHS2GVP SRFSGSGSGTDYSLTISNLDOEDIATYFCOOGNTLPYT3FGGGTKLEIK (SEQ ID NO: 15) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 16), 2=CDR2 (SEQ ID NO: 17), 3=CDR3 (SEQ ID NO: 18); (3) Light chain constant domain DNA sequence CGGGCTGATGCTGCACCA ACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTC GTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGAT GGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGA CAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGAC ATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGA GCTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 19) encoding light chain constant domain protein sequence RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSE RQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 20)B. mAb216-879Fl

[0065] Monoclonal antibody mAb216-879Fl having heavy chain and light sequences of Ighg2a_i 19464 GCTTCTTCTAACTCTAAGTTAAATCCCCTCTTGGGGTGTGAAAGCTC A CATCTCTCTCATTAGAGGTTGATCTTTGAGGAAAACAGGGTGTTGCCTAAAGGATGA AAGTGTTGAGTCTGTTGTACCTGTTGACAGCCATTCCTGGTATCCTGTCTGATGTACA GCTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTG CTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTT CCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGATGGTAGCAATAACTA CAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTT TTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAG GGGGGTACTGGGGGATATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCT CAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACA AGTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTG ACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGG CCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGA CAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCC CAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGG ATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCG AGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACA GCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGC CCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCA ACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCA GTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAA ACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGA GTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTG GACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTG GGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACC ACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGAGCTCAGCACCCACAAAAC TCTCAGGTCCAAAGAGACACCCACACTCATCTCCATGCTTCCCTTGTATAAATAAAG CACCCAGCAATGCCTGGGACCATGTAAAACTGTCCTGGTTCTTTCCAAGGTATAGAG CATAGCTCACAGGCTGATATTTCTGGCCAGGGTTGGAGGACAGCCTTGTCTATAGGA AGAGAATGAGGTTTTTGCACTGCAGGACTCAGAGCTCATTAGTTATCCTGCCTTGGA GTGTTGGGGCTTGGCTTTAGGCAGTGCCTTTTCCTTGCCTTCCTACGAACCAGCAGCT GCCATACATAGAGATAATCC (SEQ ID NO: 21) and Igk i 11233 GTCAAGACTCAGCCTG GACATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCA GATGTGATATCCAGATGACACAGACTTCATCCTCCCTGTCTGCCTCTCTGGGAGACA GAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATC AGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCACACATCAAGATTACACT CAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCA TTAGCAACCTGGATCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGC TTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCA CCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCA GTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATT GATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAAC GACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCA AGAGCTTCAACAGGAATGAGTGTTAGAGACAAAGGTCCTGAGACGCCACCACCAGC TCCCCAGCTCCATCCTATCTTCCCTTCTAAGGTCTTGGAGGCTTCCCCACAAGCGACC TACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTC CTTGGCTTTTATCATGCTAATATTTGCAGAAAATATTCAATAAAGTGAGTCTTTGCAC TTGAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 22) was developed.

[0066] A polynucleotide encoding the mAb216-879Fl heavy chain includes (1) a heavy chain variable domain signal peptide DNA sequence ATGAAAGTGTTGAGTCTGTTGTACCT GTTGACAGCCATTCCTGGTATCCTG (SEQ ID NO: 23) encoding heavy chain variable domain signal peptide MKVLSLLYLLTAIPGILS (SEQ ID NO: 24); (2) a heavy chain variable domain DNA sequence TCTGATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAAC CTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTA TTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACA TAAGCTACGATGGTAGCAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCA CTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGG ACACAGCCACATATTACTGTGCAAGGGGGGTACTGGGGGATATGGACTACTGGGGT CAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 25); a heavy chain variable domain protein sequence DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWN1WIRQFPGNKLE WMGYISYDGSNNYNPSLKN2RISITRDTSKNOFFLKLNSVTTEDTATYYCARGVLGDMD Y3WGQGTSVTVSS (SEQ ID NO: 26) with Kabat CDRs are 1 CDR1 (SEQ ID NO: 27), 2=CDR2 (SEQ ID NO: 28), 3=CDR3 (SEQ ID NO: 29); and (3) a heavy chain constant domain DNA sequence GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTG GAGATACAAGTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTG AGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCC CAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGA GCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACC AAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATG CAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAA GATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGA TGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTG GTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATG CAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCA AAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATG ACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATT TACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAAC CAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGA AGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCAC AATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGA (SEQ ID NO: 30) encoding heavy chain constant domain AKTTAPSVYPLAPVCGDTSGSSVTLGCLVKGYFPE PVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDK KIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLTPKVTCVVVDVSEDDPDVQ ISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPI ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNY KNTEPVLDSDGSYFMYSKLRVEKKNW VERNS YSC S VVHEGLHNHHTTKSF SRTPGK(SEQ ID NO: 31).

[0067] A polynucleotide encoding the mAb216-879Fl light chain includes (1) a light chain variable domain signal peptide DNA sequence ATGTCCTCTGCTCAGTTCCTTGGTCTCCTG TTGCTCTGTTTTCAAGGTACCAGATGT (SEQ ID NO: 32) encoding a light chain variable domain MSSAQFLGLLLLCFQGTRC (SEQ ID NO: 33); (2) a light chain variable domain GATATCCAGATGACACAGACTTCATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTC ACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTATCAGCAG AAACCAGATGGAACTGTTAAACTCCTGATCTACCACACATCAAGATTACACTCAGGA GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGC AACCTGGATCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCG TACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 34) encoding a light chain variable domain DIQMTQTSSSLSASLGDRVTISCRASQDISNYL N1WYOQI<PDGTVI<LLIYHTSRLHS2GVPSRFSGSGSGTDYSLTISNLDOEDIATYFCOOGN TLPYT3FGGGTKLEIK (SEQ ID NO: 35) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 36), 2=CDR2 (SEQ ID NO: 37), 3=CDR3 (SEQ ID NO: 38); (3) a light chain constant domain DNA sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGAC ATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAG TTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGT TGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAG ACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 39) encoding light chain constant domain RADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEAT HKTSTSPIVKSFNRNEC (SEQ ID NO:40).C. mAb216-878G9

[0068] Monoclonal antibody mAb216-878G9 having heavy chain and light sequences of Ighg2b_i33272 CAGATATGGACAGGCTTACTTCCTCATTCCTGCTGCTGATTGTCCCTG CATATGTCCTGTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGTCCT CCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGATAT GGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACA TTTACTGGGATGATGACACGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCT CCAAGGATACTTCTAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACACTGCAG ATACTGCCACATATAACTGTACTCGAAATCTACTATTACGACGGACGGGGGACTGGG GCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTATC CACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCTGTGACTCTGGGATGCCTGG TCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCTGTCCA GCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTACACTATGAGCAGCT CAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTC ACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCCAGCGGGCCCATTTCA ACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTC GAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATC TCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGA CGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAA CCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTGCCCTCCCCATCCAGC ACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTC CCAGCGCCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCAC TTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGG GCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTCTGACGGTTCTT ACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGAGAAAACAGATTCC TTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTACCTGAAGAAGACCAT CTCCCGGTCTCCGGGTAAATGAGCTCAGCACCCACAAAGCTCTCAGGTCCTAAGAG ACACTGGCACCCATATCCATGCATCCCTTGTATAAATAAAGCATCCAGCAAAGCCTG GTACCAT (SEQ ID NO: 41) and Igk_il270 AGCTTTGCATGGGTTCCTCCAGCCCAGCCC ACCTTCTCAGAATTTATAAACCAGGCCTTTGCATTGTGACTGATCTACATCTGAAAG GCAGGTGGAGCAAGATGGAATCACAGACTCAGGTCCTCATGTCCCTGCTGTTCTGGG TATCTGGTACCTGTGGGGACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGA CAGCAGGAGAGAAGGTCACTATGAGGTGCAAGTCCAGTCAGAGTCTGTTAAACAGT GGAGATCAAAAGAACTTCTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAA ACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGG CAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCT GGCAGTTTATTACTGTCAGAATGATTATAGTTATCCATTCACGTTCGGCTCGGGGAC AAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATC CAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTA CCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCG TCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGC ACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGC CACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTA GAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCCAGCTCCATCCTATCTTCCCTT CTAAGGTCTTGGAGGCTTCCCCACAAGCGACCTACCACTGTTGCGGTGCTCCAAACC TCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTCCTTGGCTTTTATCATGCTAATATTT GCAGAAAATATTCAATAAAGTGAGTCTTTGCACTTGAAAAAAAAAAAAAAAAAAAA AAA (SEQ ID NO: 42) was developed.

[0069] A polynucleotide encoding the mAb216-878G9 heavy chain includes (1) a heavy chain variable domain signal peptide DNA sequence ATGGACAGGCTTACTTCCTCATTCCT GCTGCTGATTGTCCCTGCATATGTCCTGTCC (SEQ ID NO: 43); a heavy chain variable domain signal peptide MDRLTSSFLLLIVPAYVLS (SEQ ID NO: 44); (2) a heavy chainvariable domain DNA sequence CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCA GTCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCT GATATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGC ACACATTTACTGGGATGATGACACGCGCTATAACCCATCCCTGAAGAGCCGGCTCAC AATCTCCAAGGATACTTCTAGAAACCAGGTATTCCTCAAGATCACCAGTGTGGACAC TGCAGATACTGCCACATATAACTGTACTCGAAATCTACTATTACGACGGACGGGGGA CTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 45) encoding a heavy chain variable domain protein sequence QVTLKESGPGILQSSQTLSLTCSFSGF SL TSDMGVS1WIROPSGI<GLEWLAHIYWDDDTRYNPSLI<S2RLTISKDTSRNOVFLI<ITS VDTADTATYNCTRNLLLRRTGD3WGOGTTLTVSS (SEQ ID NO: 46) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 47), 2=CDR2 (SEQ ID NO: 48), 3=CDR3 (SEQ ID NO: 49); and (3) a heavy chain constant domain DNA sequence GCCAAAACAACACCCC CATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCTGTGACTC TGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTG GATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTACA CTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCT GCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCCAGC GGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGCCCA GCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGAT GTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAG GATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGC TCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTGCCC TCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAAC AACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGT CAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAG ATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGT GGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGA CTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGA GAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTACCT GAAGAAGACCATCTCCCGGTCTCCGGGTAAATGA (SEQ ID NO: 50) encoding a heavy chain constant domain AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPI STINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRIS WFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER TISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDT APVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 51).

[0070] A polynucleotide encoding the mAb216-878G9 light chain includes (1) a light chain variable domain signal peptide DNA sequence ATGGAATCACAGACTCAGGT CCTCATGT CCCTGCTGTTCTGGGTATCTGGTACCTGTGGG (SEQ ID NO: 52) encoding a light chain variable domain peptide MESQTQVLMSLLFWVSGTCG (SEQ ID NO: 53); (2) a light chain variable domain sequence GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGAC AGCAGGAGAGAAGGTCACTATGAGGTGCAAGTCCAGTCAGAGTCTGTTAAACAGTG GAGATCAAAAGAACTTCTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAA CTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGC AGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTG GCAGTTTATTACTGTCAGAATGATTATAGTTATCCATTCACGTTCGGCTCGGGGACA AAGTTGGAAATAAAA (SE ID NO: 54) encoding a light chain variable domain DIVMTOSPSSLTVTAGEKVTMRCKSSOSLLNSGDOKNFLT'WYOOKPGOPPKLLIYWAS TRES2GVPDRFTGSGSGTDFTLTIS SVQAEDLAVYYCONDYSYPFT3FGSGTKLEIK (SEQ ID NO: 55) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 56), 2=CDR2 (SEQ ID NO: 57), 3=CDR3 (SEQ ID NO:58); and (3) a light chain constant domain DNA sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACA TCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAAT GTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGAC TGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCA AGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCA ACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 59) encoding a light chain constant domain RADAAPTVSIFPPSSEQLTSGGASVVCFL NNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSTSPIVKSFNRNEC (SEQ ID NO: 60)D. mAb216-878A9

[0071] Monoclonal antibody mAb216-878A9 having heavy chain and light sequences of Ighg2b_i2468 GTCTAGTTCAATGTGACTTAGGAAGCCCAGTCATATGCAAATCTAGAG AAGACTTTAGAGTAGAAATCTGAGGCTCACCTCACATACCAGCAAGGGAGTGACCA GTTAGTCTTAAGGCACCACTGAGCCCAAGTCTTAGACATCATGGATTGGGTGTGGAC CTTGCTATTCCTGATAGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGTTGGTGCA GTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTT CTGGGTATACCTTCACAGAATATCCAATGCACTGGGTGAAGCAGGCTCCAGGAAAG GGTTTCAAGTGGATGGGCATGATATACACCGACACTGGAGAGCCAACATATGCTGA AGAGTTCAAGGGACGGTTTGCCTTCTCTTTGGAGACCTCTGCCAGCACTGCCTATTT GCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGTAGCTACGGT AGGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAC CCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCTGTGA CTCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACT CTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCT ACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCA CCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGAGCCC AGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAATGC CCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAG GATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGC GAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACACAC AGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTG CCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTC AACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCT AGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGA AAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAGACATCAGTGTGG AGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTG GACTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGG GAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATTACTA CCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAATGAGCTCAGCACCCACAAAGCT CTCAGGTCCTAAGAGACACTGGCACCCATATCCATGCATCCCTTGTATAAATAAAGCATCCAGCAAAGCCTGGTACCATGTAAAAAAAAAAAAAAAAATTCAGTAATAT (SEQ ID NO: 61) and Igk_i5236 CTTTGTTAACAGACCACCTGACTTTATAAGCCAGAACTCC AAAGACTACTATTTGCATAGTTCATCCTCAGAAACCACAAATTTCTCACAGTTGTTTT AAAGAGATGCACTTATAGGAAGAGCAATAATTAGTCAGAGACCAGGATCAAAAACA CAATGGATTTTCATGTGCAGATTTTCAGCTTCATGCTAATCAGTGTCACAGTCATATT GTCCAGTGGAGAAATTGTGCTCACCCAGTCTCCAGCACTCATGGCTGCATCTCCAGG GGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTCCAGCAACTTGCA CTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTTATGGCACATCCA ACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTATTC TCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGTCAACAGTG GAGTAGTTACCCACTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTG ATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAG GTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGT GGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAG GACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGA GTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCAC CCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGAGACAAAGGTCCTGAGACGCC ACCACCAGCTCCCCAGCTCCATCCTATCTTCCCTTCTAAGGTCTTGGAGGCTTCCCCA CAAGCGACCTACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCCTTCTCCTCCT CCTCCCTTTCCTTGGCTTTTATCATGCTAATATTTGCAGAAAATATTCAATAAAGTGA GTCTTTGCACTTGAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 62) was developed.

[0072] A polynucleotide encoding the mAb216-878A9 heavy chain includes (1) a heavy chain variable domain signal peptide DNA sequence ATGGATTGGGTGTGGACCTTGCTATT CCTGATAGCAGCTGCCCAAAGTGCCCAAGCA (SEQ ID NO: 63) encoding a heavy chain variable domain signal peptide MDWVWTLLFLIAAAQSAQA (SEQ ID NO: 64); (2) a heavy chain variable domain DNA sequence CAGATCCA GTTGGTGCAGTCTGGACCTGAGCTGA AGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACA GAATATCCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTCAAGTGGATGGG CATGATATACACCGACACTGGAGAGCCAACATATGCTGAAGAGTTCAAGGGACGGT TTGCCTTCTCTTTGGAGACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAA AAATGAGGACACGGCTACATATTTCTGTGTAGCTACGGTAGGGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG (SEQ ID NO: 65) encoding a heavy chain variable domain QIQLVQSGPELKKPGETVKISCKASGYTFTEYPMH1WVKQAPGKGFKWMGMIY TDTGEPTYAEEFKG2RFAF SLET S AST AYLQINNLKNEDTAT YFC V ATVGAY3WGQGTL V TVSA (SEQ ID NO: 66) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 67), 2=CDR2 (SEQ ID NO: 68), 3=CDR3 (SEQ ID NO: 69); and (3) a heavy chain constant domain DNA sequence CCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTGGAGATACAACTG GTTCCTCTGTGACTCTGGGATGCCTGGTCAAGGGCTACTTCCCTGAGTCAGTGACTG TGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGC AGTCTGGACTCTACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAA GTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAAA AAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAG TGTCACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCT CCAAATATCAAGGATGTACTCATGATCTCCCTGACACCCAAGGTCACGTGTGTGGTG GTGGATGTGAGCGAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGT GGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTATCC GGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTC AAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAA AATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGC AGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTGGGCTTCAACCCTGGAG ACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACC GCACCAGTCCTGGACTCTGACGGTTCTTACTTCATATACAGCAAGCTCGATATAAAA ACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCT GAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAATGA (SEQ ID NO: 70) encoding a heavy chain constant domain protein sequence AKTTPPSVYPLAPGCGDT TGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQ TVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVL MISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQH QDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVV GFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRH EGLKNYYLKKTISRSPGK (SEQ ID NO: 71).

[0073] A polynucleotide encoding the mAb216-878A9 light chain includes (1) a light chain variable domain signal peptide DNA sequence ATGGATTTTCATGTGCAGATTTTCAGCTTC ATGCTAATCAGTGTCACAGTCATATTGTCCAGTGGA (SEQ ID NO: 72) encoding a light chain variable domain signal peptide MDFHVQIFSFMLISVTVILSSG (SEQ ID NO: 73); (2) a light chain variable domain sequence CTTTGTTAACAGACCACCTGACTTTATAAGC CGAGAACTCCAAAGACTACTATTTGCATAGTTCATCCTCAGAAACCACAAATTTCTC ACAGTTGTTTTAAAGAGATGCACTTATAGGAAGAGCAATAATTAGTCAGAGACCAG GATCAAAAACACAATGGATTTTCATGTGCAGATTTTCAGCTTCATGCTAATCAGTGT CACAGTCATATTGTCCAGTGGAGAAATTGTGCTCACCCAGTCTCCAGCACTCATGGC TGCATCTCCAGGGGAGAAGGTCACCATCACCTGCAGTGTCAGCTCAAGTATAAGTTC CAGCAACTTGCACTGGTACCAGCAGAAGTCAGAAACCTCCCCCAAACCCTGGATTT ATGGCACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGATCTG GGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATT ACTGTCAACAGTGGAGTAGTTACCCACTCACGTTCGGCTCGGGGACAAAGTTGGAA ATAAAA (SEQ ID NO:74) encoding a light chain variable domain EIVLTQ SP ALMAASPGEKVTITC S VS S SIS S SNLH1W YQQKSET SPKPWIYGT SNLAS2GVP VRF SGS GSGTSYSLTISSMEAEDAATYYCQQWSSYPLT3FGSGTKLEIK (SEQ ID NO: 75) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 76), 2=CDR2 (SEQ ID NO: 77), 3=CDR3 (SEQ ID NO: 78); and (3) a light chain constant domain DNA sequence CGGGCTGATGCTGCACCAA CTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCG TGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATG GCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGAC AGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACA TAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAG CTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 79) encoding a light chain constant domain RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 80).E. mAb216-961D12

[0074] Monoclonal antibody mAb216-961D12 having heavy chain and light sequences of Ighg2c_i9480 GGCATCAGCCTAGGTCCAACTCCGGAGCATGGTATAGCAGGAAGACATGCAAATAAGTCTTCTCTGTGCCCATGAAAAACACCTCGGCCCTGACCCTGCAGCTCT GACAGAGGAGGCCGGTCCTGGATTCGAGTTCCTCACATTCAGTGATGAGCACTGAA CACGGACCCCTCACCATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAA AAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCT GGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGCC ATGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATCAG TGATGGTGGTTCTTACACCTACTATCCAGACAGTGTAAAGGGCCGATTCACCGTCTC CAGAGACTATGCCATGAACAACCTGTACCTGCAAATGACCCATCTGAGGTCTGAGG ACACAGCCATGTATTTCTGTACAAGAGATGGGGGTTACAGGTACTTCGATGTCTGGG GCACAGGGACCACGGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTAT CCACTGGCCCCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTCTAGGATGCCTG GTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCC AGTGGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGCCTCTACACCCTCAGCAGC TCAGTGACTGTAACCTCGAACACCTGGCCCAGCCAGACCATCACCTGCAATGTGGCC CACCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGAGCCCAGAGTGCCCATAAC ACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCTCTT GGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTC CCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACG TCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACC CATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCAC CAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCC ATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGG TATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCT GCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGC GTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACT TCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTC GCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCC CGGTCTCTGGGTAAATGAGCTCAGCACACACAATGCTCCTGGGTCCTAATGGACACT GGCACCCATATCCATGCATCCCTTGTATAAATAAAGCACCCAGCAAAGCCTGGGAC CATGTAAAACTGTCCTGGTTCTTTCCAAGGTATAGAGCATAGCTCACGGG (SEQ ID NO: 81) and Igk_i2237 CGGGGAAATACATCAGATCAGCATGGGCATCAAGATGGAGTCACAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATT GTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGT CACCTGCAAGGCCAGCCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAAC CAGGGCAATCTCCTAAAACACTGATTTACTCGGCATCCTACCGGTTCAGTGGAGTCC CTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATG TGCAGTCTGAAGACTTGGCAGACTATTTCTGTCAACAATATAACAACTATCCATTCA CGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGGCTGATGCTGCACCAACTGTA TCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGC TTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGT GAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCAC CTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACA GCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCA ACAGGAATGAGTGTTAGAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCCAGCT CCATCCTATCTTCCCTTCTAAGGTCTTGGAGGCTTCCCCACAAGCGACCTACCACTGT TGCGGTGCTCCAAACCTCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTCCTTGGCTTT TATCATGCTAATATTTGCAGAAAATATTCAATAAAGTGAGTCTTTGCACTTGAAAA(SEQ ID NO: 82) was developed.

[0075] A polynucleotide encoding the mAb216-961D12 heavy chain includes (1) a heavy chain variable domain signal peptide DNA sequence ATGAGCACTGAACACGGACCCCTC ACCATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGT GT (SEQ ID NO: 83) encoding a heavy chain variable domain signal peptide MSTEHGPLTMNFGLSLIFLVLVLKGVQC (SEQ ID NO: 84); (2) a heavy chain variable domain DNA sequence GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAA GCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTT GCCATGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCAT CAGTGATGGTGGTTCTTACACCTACTATCCAGACAGTGTAAAGGGCCGATTCACCGT CTCCAGAGACTATGCCATGAACAACCTGTACCTGCAAATGACCCATCTGAGGTCTGA GGACACAGCCATGTATTTCTGTACAAGAGATGGGGGTTACAGGTACTTCGATGTCTG GGGCACAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 85) encoding a heavy chain variable domain EVQLVESGGGLVKPGGSLKLSCAASGFTFSSFAMS1WVRQTPEKRLEWV ATISDGGSYTYYPDSVKG2RFTVSRDYAMNNLYLOMTHLRSEDTAMYFCTRDGGYRYFDV3WGTGTTVTVSS (SEQ ID NO: 86) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 87), 2=CDR2 (SEQ ID NO: 88), 3=CDR3 (SEQ ID NO: 89); and (3) a heavy chain constant domain DNA sequence GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGG AGGTACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGA GCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCC AGCTCTCCTGCAGTCTGGCCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAA CACCTGGCCCAGCCAGACCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCA AAGTGGACAAGAAAATTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCA CTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTC ATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACA TGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTG AACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACA GTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCA AGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACC ATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCA GCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTT ACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACA AGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCA GAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCAC GAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAATGA(SEQ ID NO: 90) encoding heavy chain constant domain AKTTAPSVYPLAPVCGGTTGSSVT LGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNV AHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPM VTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMS GKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAE IAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLH NHLTTKTISRSLGK (SEQ ID NO: 91)

[0076] A polynucleotide encoding the mAb216-961D12 light chain includes (1) a light chain variable domain signal peptide DNA sequence ATGGGCATCAAGATGGAGTCACAGACTCA GGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGA (SEQ ID NO: 92) encoding a light chain variable domain peptide MGIKMESQTQVFVYMLLWLSGVDG (SEQID NO: 93); (2) a light chain variable domain sequence GACATTGTGATGACCCAGTCTCAA AAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGCCA GAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAA CACTGATTTACTCGGCATCCTACCGGTTCAGTGGAGTCCCTGATCGCTTCACAGGCA GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGG CAGACTATTTCTGTCAACAATATAACAACTATCCATTCACGTTCGGCTCGGGGACAA AGTTGGAAATAAAA (SEQ ID NO: 94) encoding a light chain variable domain D1VMTOSOKFMSTSVGDRVSVTCKASQNVGTNVA1WYOQKPGOSPI<TLIYSASYR.FS2G VPDRFTGSGSGTDFTLTISNVOSEDLADYFCOOYNNYPFT3FGSGTKLEIK (SEQ ID NO: 95) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 96), 2=CDR2 (SEQ ID NO: 97), 3=CDR3 (SEQ ID NO: 98); and (3) a light chain constant domain DNA sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACA TCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAAT GTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGAC TGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCA AGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCA ACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAG (SEQ ID NO: 99) encoding a light chain constant domain RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 100).F. mAb216-878H5

[0077] Monoclonal antibody mAb216-878H5 having heavy chain and light sequences of Ighgl i 15458 TCAGTATCCTCTTCACAGTCATTGAAAACACTGACTCTAATCATGGAT TGTAACTGGATACTTCCTTTTATTCTGTCGGTAATTTCAGGGGTCTACTCAGAGGTTC AGGTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGACTTCCGTGAAGATGTCCT GCAAGGCTTCTGGCTACAGCTTTAGCAGCTACTGGATGCACTGGGTAAAACAGAGG CCTGGACAGGGTCTAGAATGGATTGGTTCTGTTTATCCTGGAAATGGTGATACCACC TACAACCAGAGATTCAAGGACAAGGCCAAACTGACTACAGTCACATCCGCCAGCAC TGCCTACATGGTGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTTCTGTAC ATGTGATTTCGGGATCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGC CCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCC AGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGC TGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCAC CTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGG TGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCC CAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTA CTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGG TCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCC GGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACC AGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCT GCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGT GTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCT GCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGC AGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTAC TTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTT CACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTC CCACTCTCCTGGTAAATGAAAAAAA (SEQ ID NO: 101) and Igk_il233 TTTCCATATACCAGTCACATTGTGAGCCATTGTAATTGAAGTCAAGACTCAGCCTGG ACATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAG ATGTGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAG AGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCA GCAGAAACCAGATGGAACTATTCAACTCCTGATCTATCACACATCAACTCTACACTC AGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCAT CAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTGTAGTAAGGT TCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAACGGGCTGATGCTGCAC CAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAG TCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTG ATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAA GACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACG ACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGAGACAAAGGTCCTGAGACGCCACCACCAGCT CCCCAGCTCCATCCTATCTTCCCTTCTAAGGTCTTGGAGGCTTCCCCACAAGCGACCT ACCACTGTTGCGGTGCTCCAAACCTCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTC CTTGGCTTTTATCATGCTAATATTTGCAGAAAATATTCAATAAAGTGAGTCTTTGCAC TTGAAAA (SEQ ID NO: 102) was developed.

[0078] A polynucleotide encoding the mAb216-878H5 heavy chain includes (1) a heavy chain variable domain signal peptide DNA sequence ATGGATTGTAACTGGATACTTCCTT TTATTCTGTCGGTAATTTCAGGGGTCTACTCA (SEQ ID NO: 103) encoding a heavy chain variable domain signal peptide MDCNWILPFILSVISGVYS (SEQ ID NO: 104); (2) a heavy chain variable domain DNA sequence GAGGTTCAGGTCCAGCAGTCTGGGACTGTGCTGG CAAGGCCTGGGACTTCCGTGAAGATGTCCTGCAAGGCTTCTGGCTACAGCTTTAGCA GCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTAGAATGGATTGGT TCTGTTTATCCTGGAAATGGTGATACCACCTACAACCAGAGATTCAAGGACAAGGCC AAACTGACTACAGTCACATCCGCCAGCACTGCCTACATGGTGCTCAGCAGCCTGACA AATGAGGACTCTGCGGTCTATTTCTGTACATGTGATTTCGGGATCGCCTGGTTTGCTT ACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO: 105) encoding a heavy chain variable domain protein sequence EVQVQQSGTVLARPGTSVKMSCKASGYSFSSYW MH'VVVKORPGOGLEWIGSVYPGNGDTTYNORFKD2KAKLTTVTSASTAYMVLSSLTNE DSAVYFCTCDFGIAWFAY3WGQGTLVTVSA (SEQ ID NO: 106) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 107), 2=CDR2 (SEQ ID NO: 108), 3=CDR3 (SEQ ID NO: 109); and (3) a heavy chain constant domain DNA sequence GCCAAAACGACACCCCCATCTGTCTATCC ACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGT CAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAG CGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTC AGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCA CCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTA AGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGC CCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACA TCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGC ACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTC AGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAG GCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCC AAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACT GTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCAT CATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCA ACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACC ACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA (SEQ ID NO: 110) encoding a heavy chain constant domain AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFP EPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD KKIVPRDCGCKPCICTVPEVS S VFIFPPKPKDVLTITLTPKVTC VVVDISKDDPEVQF SWF VDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPI MDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 111).

[0079] A polynucleotide encoding the mAb216-878H5 light chain includes (1) a light chain variable domain signal peptide DNA sequence ATGTCCTCTGCTCAGTTCCTTGGTCTCCT GTTGCTCTGTTTTCAAGGTACCAGATGT (SEQ ID NO: 112) encoding a light chain variable domain signal peptide MSSAQFLGLLLLCFQGTRC (SEQ ID NO: 113); (2) a light chain variable domain sequence GATATCCAGATGACACAGACTACATCCTCCCTGTCT GCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAA TTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTATTCAACTCCTGATCTATCA CACATCAACTCTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGAC AGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTG TCAGCAGTGTAGTAAGGTTCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAA AA (SEQ ID NO: 114) encoding a light chain variable domain sequence DIQMTQTTSSLSASLGDRVTIS CSASOGISNYLN'WYOOKPDGTIOLLIYHTSTLHS2GVPSRFSGSGSGTDYSLTISNL PEDI ATYYCQQCSKVPYT3FGGGTKLEIK (SEQ ID NO: 115) with Kabat CDRs are 1=CDR1 (SEQ ID NO: 116), 2=CDR2 (SEQ ID NO: 117), 3=CDR3 (SEQ ID NO: 118); and (3) a light chain constant domain DNA sequence CGGGCTGATGCTGCACCAACTGTATCCATCTTC CCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACA AAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCA TGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACC TGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAAT GAGTGTTAGAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCCAGCTCCATCCTAT CTTCCCTTCTAAGGTCTTGGAGGCTTCCCCACAAGCGACCTACCACTGTTGCGGTGC TCCAAACCTCCTCCCCACCTCCTTCTCCTCCTCCTCCCTTTCCTTGGCTTTTATCATGC TAATATTTGCAGAAAATATTCAATAAAGTGAGTCTTTGCACTTGAAAA (SEQ ID NO: 119) encoding a light chain constant domain RADAAPTVSIFPPSSEQLTSGGASVVCFLN NFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC (SEQ ID NO: 120).

[0080] Modifications and / or changes may be made in the amino acid composition of polypeptides (e.g., antibody or antibody fragments), and thus the present invention contemplates variation in sequences of the polypeptides, and nucleic acids coding therefore, where they are nonetheless able retain substantial activity with respect to the preparative, therapeutic, preventative, and curative aspects of the present invention.

[0081] A biological functional equivalent may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the “parent” polypeptide(e.g., antibody or antibody fragments). This can be accomplished through the degeneracy of the genetic code. In one example, one of skill in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide while not disturbing the ability of that polynucleotide to encode a protein. In another example, a polynucleotide may encode a biological functional equivalent of an antibody or antibody fragment with more significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like. So-called “conservative” changes do not disrupt the biological activity of the polypeptide, as the structural change is not one that impinges on the polypeptide's ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.

[0082] In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” polypeptide and / or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) in selected amino acids (or nucleotides) may be substituted. In certain aspects, a polynucleotide or polypeptide is or is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:1, 2, 5, 6, 14, 15, 21, 22, 25, 26, 34, 35, 41, 42, 45, 46, 54, 55, 61, 62, 65, 66, 74, 75, 81, 82, 85, 86, 94, 95, 101, 102, 105, 106, 114, or 115.

[0083] In general, the shorter the length of the molecule, the fewer changes that can be made within the molecule while retaining function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification. Function of a polypeptide can be determined by using various assays known to detect the activity of the polypeptide of interest.

[0084] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and / or the like. An analysis of the size, shape and / or type of the amino acid side-chain substituents reveals that arginine, lysine, and / or histidine are all positively charged residues; that alanine, glycine, and / or serine are all a similar size; and / or that phenylalanine, tryptophan, and / or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined herein as biologically functional equivalents.

[0085] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and / or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and / or arginine (-4.5).

[0086] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index and / or score and / or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0087] It also is understood that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0088] The antibodies or functional fragments thereof (antibody fragments) of the disclosed subject matter can be generated from or adapted to any species. The antibodies or functional fragments thereof described herein can be labeled or otherwise conjugated to various chemical or biomolecule moieties, for example, for isolation, or therapeutic or diagnostic or detection or treatment applications. The moieties can be cytotoxic, for example, bacterial toxins, viral toxins, radioisotopes, and the like. The moieties can be detectable labels, for example, fluorescent labels, radiolabels, biotin, and the like, which are known in the art. The moieties can be purification tags that can be conjugated to antibodies for various applications. Purification tags include but are not limited to FLAG-tag, His-tag (Poly-Histidine tag), HA-tag (Hemagglutinin tag), Myc-tag, S-tag, GST-tag (Glutathione S-transferase), MBP-tag (Maltose-binding protein), Strep-tag, CBP-tag (Calmodulin Binding Peptide), T7-tag, V5-tag, Spot-tag, or an NE-tag. These tags facilitate the purification, detection, and study of recombinant proteins, including antibodies, by providing specific binding sites for various resins or antibodies.

[0089] In certain aspects, when an antibody or antibody fragment is used, for flow cytometric detection, for scanning laser cytometric detection, or for fluorescent immunoassay,they can be labeled with fluorophores or particles / beads, e ., magnetic or superparamagnetic polymer particles. There are a wide variety of fluorophore labels that can be attached to the antibodies or peptides of the present invention. For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the present invention can usefully be labeled with biotin. When the antibodies or antibody fragments of the present invention are used.

[0090] In light of the current specification, the determination of an appropriate use for the antibodies or antibody fragments is within the skill of the art. The components described herein will be formulated in association with an acceptable carrier. Such vehicles are usually nontoxic and non-therapeutic. The acceptable excipients described herein, for example, vehicles, adjuvants, carriers, or diluents, are well known and readily available.III. Kits

[0091] In another aspect, the present invention provides kits for performing sex selection of sperm. In one embodiment, the kit comprises one or more antibody or antibody fragment described herein with an optional solid support, such as a chip, a microtiter plate, a bead, or a resin having an antibody or antibody fragment attached (captured), wherein the captured antibody or antibody fragment binds a targeted sperm. In the case of antibody reagents, the kit can comprise a solid support with a reactive surface, and a container comprising the antibody capture reagent. The kit can also comprise a washing solution or instructions for making a washing solution, in which the combination of the antibody reagent and the washing solution allows sex selection of semen. The kit may include more than one type of adsorbent, each present on a different solid support.

[0092] In a further embodiment, such a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. For example, the instructions may inform a consumer about how to collect the sample, how to wash the probe or the particular biomarkers to be detected. In yet another embodiment, the kit can comprise one or more containers with biomarker samples, to be used as standard(s) for calibration.IV. Examples

[0093] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that thetechniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0094] Example 1 - Sex Skewing in Porcine Semen

[0095] The methods described herein for sex skewing of bovine semen were applied to porcine semen samples obtained from three boars. Specifically, the same Gx-based separation techniques were used to isolate Gx 1 positive sperm cells.

[0096] In the Gx 1 positive fraction, approximately 74% of the sperm were female (XX)-bearing. Post-skewing evaluation showed that the sorted sperm exhibited normal motility and morphology, comparable to results previously observed in bovine sperm.

[0097] Note that the porcine semen samples were not subjected to cry opreservation, as porcine semen is known to be particularly sensitive to freezing processes. Additionally, these preliminary results were obtained using semen from only three boars.

[0098] These findings indicate that the Gx marker and associated separation methods are conserved between porcine and bovine species, displaying similar functional characteristics in porcine semen as reported for bovine semen.

[0099] Example 2 - In Vitro Sex Skewing in Bovine Semen

[0100] Bovine semen samples are processed in vitro using anti-GXl monoclonal antibodies conjugated to magnetic particles, styrofoam beads, or mesh supports. Incubation allows binding to GX1 -positive X-sperm. Separation via magnetic field, filtration, or centrifugation yields X-enriched (bound) and Y-enriched (unbound) fractions. Preliminary results show skewing toward female offspring (X-enriched fractions), with motility and morphology comparable to unsorted controls. Optimization addresses minor cell losses (18-20%) through negative selection strategies.

Claims

CLAIMS1. A monoclonal antibody or antibody fragment that selectively binds to GX1, said antibody comprising:(i) heavy chain CDRs as set forth in SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69 and light chain CDRs as set forth in SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78; (ii) heavy chain CDRs as set forth in SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49 and light chain CDRs as set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58; (iii) heavy chain CDRs as set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 and light chain CDRs as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18;(iv) heavy chain CDRs as set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109 and light chain CDRs as set forth in SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO:118;(v) heavy chain CDRs as set forth in SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 and light chain CDRs as set forth in SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; or (vi) heavy chain CDRs as set forth in SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89 and light chain CDRs as set forth in SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:

98.

2. The monoclonal antibody or antibody fragment of claim 1, wherein an antibody or antibody fragment comprise:(i) a heavy chain variable region that is at least 90% identical to SEQ ID NO:66 and light chain variable region that is at least 90% identical to SEQ ID NO:75;(ii) a heavy chain variable region that is at least 90% identical to SEQ ID NO:46 and light chain variable region that is at least 90% identical to SEQ ID NO:55;(iii) a heavy chain variable region that is at least 90% identical to SEQ ID NO:6 and light chain variable region that is at least 90% identical to SEQ ID NO: 15;(iv) heavy chain variable region that is at least 90% identical to SEQ ID NO: 106 and light chain variable region that is at least 90% identical to SEQ ID NO: 115;(v) a heavy chain variable region that is at least 90% identical SEQ ID NO: 26 and light chain variable region that is at least 90% identical SEQ ID NO:35; or(vi) a heavy chain variable region that is at least 90% identical SEQ ID NO:86 and light chain variable region that is at least 90% identical SEQ ID NO:95.

3. The monoclonal antibody or antibody fragment of claim 1 , further comprising a heterologous moiety.

4. The monoclonal antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is coupled to a polymeric or magnetic particle.

5. A method of selecting sex specific sperm comprising:(i) contacting a sperm composition with one or more antibody or antibody fragment of claim 1 forming an X chromosome containing sperm complex and a non-complexed Y chromosome containing sperm; and(ii) isolating the X chromosome containing sperm complex to form an enhanced X chromosome containing sperm composition and / or isolating the non-complexed Y chromosome containing sperm to form an enhanced non-complexed Y chromosome containing sperm composition.

6. The method of claim 5, further comprising evaluating the enhanced X chromosome containing sperm composition and / or the enhanced non-complexed Y chromosome containing sperm composition for motility, morphology, or motility and morphology.

7. The method of claim 5, further comprising preserving the enhanced X chromosome containing sperm composition and / or the enhanced non-complexed Y chromosome containing sperm composition.

8. The method of claim 7, wherein the enhanced X chromosome containing sperm composition and / or the enhanced non-complexed Y chromosome containing sperm composition are frozen.

9. A method of inseminating a mammal comprising depositing an enhanced X chromosome containing sperm composition or an enhanced non-complexed Y chromosome containing sperm composition into a uterus of a female mammal.

10. A method of fertilizing a mammalian egg comprising contacting an egg in vitro with an enhanced X chromosome containing sperm composition or an enhanced non-complexed Y chromosome containing sperm composition forming a fertilized egg.

11. The method of claim 10, further comprising implanting the fertilized egg in a recipient female.

12. The monoclonal antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is a Fab, Fab', F(ab')2, scFv, or single-domain antibody.

13. The monoclonal antibody or antibody fragment of claim 1 , wherein the antibody or antibody fragment is coupled to a non-magnetic particle.

14. The monoclonal antibody or antibody fragment of claim 13, wherein the non-magnetic particle is a polymeric bead, styrofoam bead, or porous bead.

15. The monoclonal antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is coupled to a mesh, fdter, or column support.

16. A composition comprising two or more monoclonal antibodies or antibody fragments of claim 1, wherein each antibody or fragment comprises a different set of heavy and light chain CDRs selected from (i) through (vi).

17. The method of claim 5, wherein the one or more antibody or antibody fragment is coupled to a magnetic particle, and isolating comprises applying a magnetic field (positive selection).

18. The method of claim 5, wherein isolating the enhanced non-complexed Y chromosome containing sperm composition comprises negative selection by depleting X chromosome containing sperm complexes.

19. The method of claim 5, wherein the one or more antibody or antibody fragment is coupled to a non-magnetic particle, styrofoam bead, porous bead, mesh, or filter, and isolating comprises filtration, centrifugation, or column chromatography.

20. The method of claim 5, wherein the antibody or antibody fragment is a Fab, scFv, or antibody fragment that reduces steric hindrance on bound sperm.

21. The method of claim 5, resulting in an enhanced X chromosome containing sperm composition with at least 70% X chromosome bearing sperm and / or an enhanced Y chromosome containing sperm composition with at least 70% Y chromosome bearing sperm.

22. The method of claim 5, wherein total sperm recovery is at least 80%, or wherein cell loss in the bound fraction is about 18-20%.

23. The method of claim 5, performed on bovine or porcine sperm.

24. A kit for selecting sex-specific sperm comprising: one or more monoclonal antibodies or antibody fragments of claim 1; and a solid support selected from a magnetic particle, polymeric bead, styrofoam bead, mesh, or filter.

25. The kit of claim 24, further comprising instructions for positive selection, negative selection, or both.

26. The method of claim 9, wherein the mammal is bovine or porcine.

27. The method of claim 5, further comprising pooling two or more antibodies or fragments having different CDR sets from claim 1.

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