Anti-izumo1 immunoglobulins and methods of use thereof

Abstract

An immunoglobulin that binds an IZUMO protein, the immunoglobulin comprising a heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2, and CDR- H3, including for example wherein the amino acid sequences of said CDRs are SEQ ID NOs: 1- 3; SEQ ID NOs: 1, 4, and 5; SEQ ID NOs: 1, 6, and 7; SEQ ID NOs: 1, 8, and 9; SEQ ID NOs: 1, 10, and 11; SEQ ID NOs: 1, 12, and 13; SEQ ID NOs: 1, 14, and 15; SEQ ID NOs: 16-18; SEQ ID NOs: 1, 19, and 20; SEQ ID NOs: 21-23; SEQ ID NOs: 24-26; SEQ ID NOs: 27-29; SEQ ID NOs: 59-61; SEQ ID NOs: 62-64; or SEQ ID NOs: 1, and 65-66. Nucleic acids, vectors, cells, compositions, and methods of use thereof are also described.

Description

TITLE: ANTI-IZUM01 IMMUNOGLOBULINS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to Canadian Application No. 3,215,576 filed October 6, 2023, the contents of which is incorporated herein by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “30344- P73343PC00_Sequence_Listing” (81 ,300 bytes) created on October 7, 2024, is herein incorporated by reference.FIELD

[0003] The present disclosure relates to the development of immunoglobulins, e.g., nanobodies, to IZUMO1 and the use of these immunoglobulins for inhibiting interaction of IZUMO1 protein with for example JUNO protein and / or oocytes, and in particular for inhibiting binding of sperm with an oocyte, for use as non-hormonal contraceptives.BACKGROUND

[0004] There are a number of forms of contraception available, including the condom, birth control pill, the vaginal ring, copper IUD, the contraceptive skin patch and hormone-releasing contraceptive coils. But many of these options have issues that prevent them from being widely adopted.

[0005] Hormonal contraceptives have potential side effects such as headaches, nausea, sore breasts and vaginal yeast infections (thrush). The hormones can also cause spotting between periods or lead to mood swings and may reduce women’s sexual desire. With contraceptives, this is a highly personal choice. Some users prefer hormonal contraceptives, others prefer condom, some prefer an implant, others a pill or injectable. Thus, it is important to develop new effective non- hormonal contraceptives to increase the options for users.

[0006] As of 2019, more than half of the 1.6 billion women of reproductive age (15-49 years) living in low and middle income countries (LMICs) want to avoid a pregnancy. Globally, approximately 257 million women had an unmet need for modern contraception — that is, they want to avoid a pregnancy but were not using a modern method. About half (49%) of pregnancies in LMICs — 111 million annually — are unintended pregnancies, the majority (77%) occur among women who want to avoid a pregnancy but are not using modern contraceptives. Theseunintended pregnancies result in an estimated 30 million unplanned births and 69 million induced abortions.

[0007] Although existing modern contraceptive methods are highly effective and safe for most women, they have features that limit uptake and continued use for some, including requirements for a skilled provider for administration, routes of administration that lack discretion, and side effects that cause dissatisfaction and subsequent incorrect use or discontinuation. Despite this, innovation leading to new contraceptive products suitable for use in LMICs has been limited. Much of the product development that has occurred has been innovations in delivery of the same classes of hormones, which cause similar side effects and do not fully address women’s underlying issues and concerns.

[0008] Many women with unmet contraceptive needs have expressed interest in using products that differ from currently available methods and that particularly address key barriers they face in accessing and using contraceptives, including expressed interest in easy-to-access, discreet, convenient, non-invasive methods of contraception with more tolerable side effects, in particular no impact on the normal menstrual cycle.SUMMARY

[0009] Provided herein are immunoglobulins, such as nanobodies, that can be used for inhibiting binding of JUNO proteins with IZUMO1 proteins, proteins which are involved in spermegg attachment and fusion during fertilization.

[0010] A key step in fertilization is the fusion of the egg and sperm plasma membranes, which allows the merger of the two gametes. Several sperm / egg proteins are essential for fusion: egg (JUNO, MAIA and CD9); sperm (IZUMO1, SPACA6, SOF1 , TMEM95, DCST-1 , DCST-2, TMEM81 and FIMP). Egg JUNO and sperm IZUMO1 are two important gamete proteins involved in fertilization. Both are expressed primarily on gametes and their interaction has been proven to be involved in egg and sperm fusion. The interaction between JUNO and IZUMO1 is demonstrated herein to be blocked by the nanobodies described herein (camelid single-domain antibodies), providing a non-hormonal method of contraception. There is a need for products with different features and properties. For example, there may be an array of preferences for route of administration, duration of contraception, and ability for self-administration. The versatility of nanobodies allows flexibility for drug delivery. Due to the nanobody’s high solubility and stability, they can be developed as an injectable for systemic delivery, and / or intravaginal local drug delivery via a dissolvable vaginal film, intrauterine device, ring or gel.

[0011] The immunoglobulins described herein are not based on hormones, thus likely having less side-effects than current hormonal contraceptive therapies. Nanobodies are stable molecules that can be prepared in multiple modes of formulation (e.g., injectable for continuous systemic delivery or gel for topical / local delivery or vaginal film / ring for continuous local delivery).

[0012] Accordingly, an aspect of the disclosure includes an immunoglobulin that binds an IZIIMO protein, the immunoglobulin comprising a heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3 and optionally a signal peptide, wherein the amino acid sequences of said CDRs are: a)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 2); andWVSTINNDGSIANCDR-H3 (SEQ ID NO: 3);TIGIKNTWRPCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 4); andWVSAITSGGGSTTCDR-H3 (SEQ ID NO: 5);APGTYHSGPIPD c)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 6); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 7);APGESPAFDGLVEVMD d)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 8); andWVSTINSGGGSTSCDR-H3 (SEQ ID NO: 9); orAPESATSAQAMANIPDCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 10); andWVSAINSDGGSTSCDR-H3 (SEQ ID NO: 11);APGRDSDYDIIPD f)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 12); andWVSIINSGGASTYCDR-H3 (SEQ ID NO: 13);APEGNPGPIATIPD g)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 14); andWVSTINSGGGKTSCDR-H3 (SEQ ID NO: 15);EPGYQRRYEYD h)CDR-H1 (SEQ ID NO: 16);SSYVMGCDR-H2 (SEQ ID NO: 17); andWVSTITSDGTSARCDR-H3 (SEQ ID NO: 18);APEEKIPIASIPE i)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 19); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 20);APGDSREHGSTWYAI PD j)CDR-H1 (SEQ ID NO: 21);SDYAMTCDR-H2 (SEQ ID NO: 22); andWVSSISSGGGSTRCDR-H3 (SEQ ID NO: 23).DIGWTFNNL k)CDR-H1 (SEQ ID NO: 24);SINTMGCDR-H2 (SEQ ID NO: 25); andLVAIINSGDRTNCDR-H3 (SEQ ID NO: 26); orNVDSPLYPGRG l)CDR-H1 (SEQ ID NO: 27);SINVMGCDR-H2 (SEQ ID NO: 28); andLVAEITSGDTTNCDR-H3 (SEQ ID NO: 29)NIDSPRYGP m)CDR-H1 (SEQ ID NO: 59);SISAMTCDR-H2 (SEQ ID NO: 60); andWVARISSGGTTHCDR-H3 (SEQ ID NO: 61);NGIALTEANGSRRYNA n)CDR-H1 (SEQ ID NO: 62);SVNGMGCDR-H2 (SEQ ID NO: 63); andLVARITSGSYTDCDR-H3 (SEQ ID NO: 64);WYTDYVDNDGVQ o)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 65); andWVSTINNGGGSTSCDR-H3 (SEQ ID NO: 66); orAPGDYSDYVLQPDFGP)CDR-H1 (SEQ ID NO: 82);SVNGMGCDR-H2 (SEQ ID NO: 83); andMVARMTTGGCDR-H3YYLDFVGGEGGSLE(SEQ ID N0: 84)'

[0013] Another aspect of the disclosure includes an immunoglobulin which competes with the immunoglobulin of any one of claims 1 to 25 for binding to an IZIIMO protein.

[0014] Another aspect of the disclosures includes a nucleic acid molecule encoding any immunoglobulin described herein.

[0015] Another aspect of the disclosures includes a vector comprising the nucleic acid molecule described herein.

[0016] Another aspect of the disclosures includes a cell comprising any nucleic acid molecule, any vector, or expressing any immunoglobulin described herein.

[0017] Another aspect of the disclosures includes a composition comprising any immunoglobulin, any nucleic acid molecule, any vector, or any cell of claim described herein, and a diluent or pharmaceutically acceptable carrier.

[0018] Another aspect of the disclosures includes a method of inhibiting binding of a JUNO protein with an IZUMO1 protein, the method comprising contacting the IZUMO1 protein with any immunoglobulin described herein.

[0019] Another aspect of the disclosures includes a method of inhibiting fertilization, the method comprising contacting a IZUMO1 protein with any immunoglobulin described herein.

[0020] Another aspect of the disclosures includes use of any immunoglobulin described herein, or any composition described herein as a contraceptive.

[0021] Another aspect of the disclosures includes a protein complex comprising an IZUMO1 protein interacting with any immunoglobulin described herein.

[0022] Another aspect of the disclosures includes a method of making any immunoglobulin described herein comprising: a) introducing any nucleic acid described herein or any vector described herein, in a suitable cell or b) culturing any cell described herein; and isolating the immunoglobulin.

[0023] Another aspect of the disclosure includes a kit or immunological assay comprising one or more the immunoglobulin described herein, the nucleic acid molecule described herein,the vector described herein, the cell described herein, and / or the composition described herein, and optionally a container or vial.

[0024] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0025] An embodiment of the present disclosure will now be described in relation to the drawings in which:

[0026] Figs. 1A-1C: Hamster oocyte / human sperm in vitro fertilization functional assays of anti-IZUMO1 nanobodies. Nanobodies that have better than 90% IVF inhibition are selected for further development (Nb3-25545, Nb6-25619, Nb10-25648, Nb1-25600, Nb4-25568, Nb5- 25557, Nb8-25553). Number above bars refer to the number of oocyte replicates performed.

[0027] Fig. 2: Schematic design of periplasmic pET-22b vector for Anti-IZUMO1 expression periplasmic expression

[0028] Figs. 3A-B: A) SDS-PAGE of Anti-IZUMO1 after IMAC and size chromatography purification. 16% polyacrylamide gel, tris-glycine buffer, run for 20 minutes at 120 V then for 50 minutes at 200 V. Lane 1 : Flow-thru, Lane 2: Wash, Lane 3: Eluted of Anti-IZUMO1 with His-T ag, Lane 4: BLUEstain™ protein Ladder, Lane 5: Eluted Anti-IZUMO1 with His-Tag, Lane 6: Anti- IZUMO1 after thrombin digestion and size exclusion chromatography. B) Size Exclusion chromatogram of IMAC-purified and thrombin-digested Anti-IZUMO1 nanobody.

[0029] Fig. 4: Representative MALDI-TOF spectra of Anti-IZUMO1 with and without 10xHis: The nanobody was best detected in Linear positive mode: MALDI-TOF-MS: m / z calculated for the nanobody with His-Tag [M-H]+ 15608.26 Da, found 15506.484 Da. MALDI- TOF-MS: m / z calculated for the nanobody without His-Tag [M-H]+ 14092.71 Da, found 13957.774 Da. The difference of the found masses (Am / z: 1548.712 Da) reflects the mass of the cleaved 10xHis.

[0030] Figs. 5A-D: Characterization of thermal unfolding of Anti-IZUMO1 nanobodies. The inflection temperatures (Ti) are obtained, a) #4 Anti-IZUMO1 with a Ti = 63.5 ± 0.96 °C, b) #5 Anti-IZUMO1 with a Ti = 62.1 ± 1.03 °C. c) #6 Anti-IZUMO1 with a Ti = 56.9 ± 0.85 °C. d) #8 Anti- IZUMO1 with a Ti = 62.9 ± 0.97 °C.

[0031] Figs. 6A-L: Representatives binding kinetics analysis of the interaction between Anti-IZUMO1 nanobodies and human IZLIMO1 using biolayer interferometry (BLI). Human IZLIMO1 was biotinylated and immobilized onto streptavidin-coated bionsensors. The binding kinetics parameters were obtained by fitting the data to 1 :1 binding model. The fitted curves are shown in lighter grey lines superimposed to the experimental curves showcased in darker grey.

[0032] Fig. 7: Representatives half inhibitory concentration (IC50) measurements of the Anti-IZUMO1 nanobodies to reflect the inhibition of the interaction between IZLIMO1 and JUNO. Assay mixture included 200 nM of biotinylated IZUMO1 , 500 nM of JUNO and varying concentrations of Anti-IZUMO1 nanobodies. Assays were conducted at room temperature. The results are expressed as mean ± standard error of the mean (n = 3).

[0033] Fig. 8: Binding affinity of IZUMO1 nanobodies to IZUMO1 after thermal and chemical stress tests, p: Sample precipitated after stress test.

[0034] Fig. 9: Aggregation temperature of IZUMO1 after thermal and chemical stress tests. *: Tagg cannot be determined because the sample is aggregated before thermal ramp, p: Sample precipitated after stress test.

[0035] Fig. 10: Crystallization and data collection of ZNb11-IZUMO1. Crystals of degycosylated IZUMO1 Tag Off and ZNb11 Tag Off complex that grew in the further optimized condition (1.5 M Ammonium sulfate, 0.1 M BisTris pH 5.5, 1% w / v PEG 3350, 1X silver bullet) and diffracted to 3 A. Data collection statistics are shown in the Table 9.

[0036] Fig. 11 : Schematic representation of the IZUMO1-JUNO crystal structure (left) and IZUMO1-ZNb11 co-complex (right). ZNb11 binds with a direct overlap of the JUNO binding site.

[0037] Fig. 12: Molecular surface representation of IZUMO1-JUNO and ZNb11-IZUMO1 interface. The binding footprint of JUNO (left) and ZNb11 (right) are shown. ZNb11 has a 77.3% overlap with JUNO binding site.DETAILED DESCRIPTIONDefinitions

[0038] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0039] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For example, the term "a cell" includes a single cell as well as a plurality or population of cells. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art (see, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York.).

[0040] Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0041] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds.

[0042] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0043] As used in this application and claim(s), the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements,components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0044] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, (such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0045] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0046] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0047] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of" or, when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0048] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0049] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0050] The term “complementarity determining region” or “CDR” as used herein refers to particular hypervariable regions of immunoglobulins that are commonly presumed to contribute to epitope binding. Computational methods for identifying CDR sequences in nanobodies include .. A person skilled in the art having regard to the sequences comprised herein would also be able to identify CDR sequences based on different CDR calling programs. Such immunoglobulins are similarly encompassed. Various programs may be used to identify CDRs. CDRs for variable domains for any CDR calling programs are encompassed.

[0051] The term "immunoglobulin" as used herein is intended to encompass for example immunoglobulin binding proteins having at least a heavy chain variable domain (e.g., a nanobody or VHH or heavy chain antibody such as a camelid antibody). The term “heavy chain variable domain” refers to a domain having 4 framework regions (FR) and 3 CDRs in the orientation of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.The binding proteins may be from recombinant sources and / or produced in transgenic animals. They can for example be produced using biochemical techniques, or can be isolated from a library such as a phage display library. Binding protein backbones may comprise any suitable variable heavy chain and / or additional immunoglobulin domains (e.g., Fc domain). Binding proteins, including humanized and / or other chimeric binding proteins may include sequences from one or more than one isotype, class, or species. Immunoglobulins binding proteins may be any class of immunoglobulins including: IgG, IgM, IgD, IgA, or IgE; and any isotype thereof, including lgG1 , lgG2 (e.g. lgG2a, lgG2b, lgG2c), lgG3 and lgG4.

[0052] The term “nanobody” and variations thereof (e.g., VHH etc), refer to antigenbinding polypeptides that can be fragments of camelid antibodies and that are or comprise a heavy chain variable region including CDRH1 , CDRH2, and CDRH3 and include what is referred to as the VHH domain from camelid heavy chain antibodies. Nanobodies described herein may be described with reference to an identifier such as ZNb followed by a number or Nb followed by a number. Where the first number is the same, they refer to the same or related sequences, (e.g., ZNb4 and Nb4 and Nb4-25568 refer to the same nanobody).

[0053] Compositions of the invention may comprise an effective amount of a compound in combination with a conventional pharmaceutical carrier. As used herein the term “pharmaceutically acceptable carrier” means a solid or liquid filler, diluent or encapsulating material. See, for example, U.S. Pat. No. 5,580,852, herein incorporated by reference.

[0054] The term “cell” as used herein refers to a single cell or a plurality of cells.

[0055] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative substitution” also includes the use of a chemically derivatized residue or non-natural amino acid in place of a non-derivatized residue provided that such polypeptide displays the requisite activity.

[0056] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to any chain of two or more natural or unnatural amino acid residues, regardless of post-translational modifications (e.g., glycosylation or phosphorylation). Included are proteins that are a single polypeptide chain and multisubunit proteins (e.g., composed of 2 or more polypeptides).

[0057] The term "sequence identity" as used herein refers to the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequencesare aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , % identity = [number of identical overlapping positions] I [total number of positions] X 100%). The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBI_AST programs of Altschul et al., 1990. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g. for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g. to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. of XBLAST and NBLAST) can be used (see, e.g. the NCBI website). Another nonlimiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0058] For immunoglobulins, percentage sequence identities can be determined when immunoglobulin sequences are maximally aligned by Kabat, IMGT, or other numbering conventions. The terms “Kabat numbering”, ”IMGT numbering”, etc., which are recognized in theart, refer to systems of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and / or light chain variable regions of an immunoglobulin, or antigen binding portion thereof. After alignment, if a subject immunoglobulin region (e.g., the entire mature variable region of a heavy) is being compared with the same region of a reference immunoglobulin, the percentage sequence identity between the subject and reference immunoglobulin regions is the number of positions occupied by the same amino acid in both the subject and reference immunoglobulin region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage. Accordingly, Kabat, IMGT, and other alignment systems can also be used to identify or annotate CDRs in an immunoglobulin sequence.

[0059] The term "nucleic acid” or “nucleic acid molecule", as used herein, are intended to include unmodified DNA or RNA or modified DNA or RNA. The nucleic acid molecules of the disclosure may contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. Unless otherwise indicated, standard IUPAC-IUB nomenclature is used herein. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid molecule" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The term "isolated nucleic acid molecules" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.

[0060] The term "vector" as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, Adeno Associated viral vectors and the like.

[0061] The term “pharmaceutically acceptable” means compatible with administration to animals, in particular, humans.

[0062] The term "administered" as used herein means administration of a therapeutically effective dose of an immunoglobulin, immunoconjugate, or composition of the disclosure to a cell or subject.

[0063] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.

[0064] The term “bispecific construct” or similar terms, as used herein refer to an immunoglobulin that is a construct in which two heavy chain variable domains (e.g., nanobodies) which bind the same or an overlapping epitope sequence (e.g., including discontinuous 3D epitope sequences composed of residues from various parts of the protein) on the same antigen (e.g., IZLIMO1 protein) are linked directly or indirectly e.g., by a linker. A bispecific construct can for example bind two molecules of IZLIMO1 protein.

[0065] The term “multi-specific construct” or similar terms, as used herein refer to an immunoglobulin that is a construct in which more than two heavy chain variable domains (e.g., nanobodies) which bind the same or an overlapping epitope sequence (e.g., including discontinuous 3D epitope sequences composed of residues from various parts of the protein) on the same antigen (e.g., IZLIMO1 protein) are linked directly or indirectly e.g., by linkers. A multispecific construct can for example bind two or more molecules of IZLIMO1 protein or the same IZLIMO1 protein.

[0066] The term “bi-paratopic construct” or similar terms, as used herein refer to an immunoglobulin that is a construct in which two heavy chain variable domains (e.g., nanobodies) which bind different epitopes on the same antigen (e.g., IZLIMO1 protein) are linked directly or indirectly e.g., by a linker. A biparatropic construct can for example bind the same molecule or two molecules of IZLIMO1 protein.

[0067] The term “multi-valent construct” or derivatives thereof, as used herein refer to an immunoglobulin that is a construct in which more than two heavy chain variable domains (e.g., nanobodies) which bind different epitopes on the same antigen (e.g., IZLIMO1 protein) are linked directly or indirectly e.g., by linkers. A multi-valent construct can for example bind two or more molecules of IZLIMO1 protein or the same IZLIMO1 protein.

[0068] The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.

[0069] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about".

[0070] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0071] The immunoglobulins described herein can be used in continuous use non- hormonal contraceptives, optionally female contraceptives, that significantly differ from currently available products with a particular aim of not disrupting endogenous menstrual bleeding patterns since this is a common concern raised by users who discontinue and / or avoid use of hormonal contraceptives.

[0072] An aspect of the disclosure includes an immunoglobulin that binds a IZLIMO1 protein, the immunoglobulin comprising a heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3 and optionally a signal peptide, wherein the amino acid sequences of said CDRs are as set forth in Table 5.

[0073] In some embodiments, the CDRs are SEQ ID NOs: 1-3. In some embodiments, the CDRs are SEQ ID NOs: 1 and 4-5. In some embodiments, the CDRs are SEQ ID NOs: 1 and 6-7. In some embodiments, the CDRs are SEQ ID NOs: 1 and 8-9. In some embodiments, the CDRs are SEQ ID NOs: 1 and 10-11. In some embodiments, the CDRs are SEQ ID NOs: 1 and 12-13. In some embodiments, the CDRs are SEQ ID NOs: 1 and 14-15. In some embodiments, the CDRs are SEQ ID NOs: 16-18. In some embodiments, the CDRs are SEQ ID NOs: 1 and 19- 20. In some embodiments, the CDRs are SEQ ID NOs: 21-23. In some embodiments, the CDRs are SEQ ID NOs: 24-26. In some embodiments, the CDRs are SEQ ID NOs: 27-29.

[0074] In some embodiments, the immunoglobulin comprises a heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3 and optionally a signal peptide, wherein the amino acid sequences of said CDRs are:CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 2); andWVSTINNDGSIANCDR-H3 (SEQ ID NO: 3);TIGIKNTWRPCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 4); andWVSAITSGGGSTTCDR-H3 (SEQ ID NO: 5);APGTYHSGPIPD c)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 6); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 7);APGESPAFDGLVEVMD d)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 8); andWVSTINSGGGSTSCDR-H3 (SEQ ID NO: 9); orAPESATSAQAMANIPD e)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 10); andWVSAINSDGGSTSCDR-H3 (SEQ ID NO: 11);APGRDSDYDIIPD f)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 12); andWVSIINSGGASTYCDR-H3 (SEQ ID NO: 13);APEGNPGPIATIPDCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 14); andWVSTINSGGGKTSCDR-H3 (SEQ ID NO: 15);EPGYQRRYEYD h)CDR-H1 (SEQ ID NO: 16);SSYVMGCDR-H2 (SEQ ID NO: 17); andWVSTITSDGTSARCDR-H3 (SEQ ID NO: 18);APEEKIPIASIPE i)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 19); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 20);APGDSREHGSTWYAI PD j)CDR-H1 (SEQ ID NO: 21);SDYAMTCDR-H2 (SEQ ID NO: 22); andWVSSISSGGGSTRCDR-H3 (SEQ ID NO: 23).DIGWTFNNL k)CDR-H1 (SEQ ID NO: 24);SINTMGCDR-H2 (SEQ ID NO: 25); andLVAIINSGDRTNCDR-H3 (SEQ ID NO: 26); orNVDSPLYPGRG l)CDR-H1 (SEQ ID NO: 27);SINVMGCDR-H2 (SEQ ID NO: 28); andLVAEITSGDTTNCDR-H3 (SEQ ID NO: 29)NIDSPRYGPm)CDR-H1 (SEQ ID NO: 59);SISAMTCDR-H2 (SEQ ID NO: 60); andWVARISSGGTTHCDR-H3 (SEQ ID NO: 61);NGIALTEANGSRRYNA n)CDR-H1 (SEQ ID NO: 62);SVNGMGCDR-H2 (SEQ ID NO: 63); andLVARITSGSYTDCDR-H3 (SEQ ID NO: 64);WYTDYVDNDGVQ o)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 65); andWVSTINNGGGSTSCDR-H3 (SEQ ID NO: 66); orAPGDYSDYVLQPDFGP)CDR-H1 (SEQ ID NO: 82);SVNGMGCDR-H2 (SEQ ID NO: 83); andMVARMTTGGCDR-H3 (SEQ ID NO: 84).YYLDFVGGEGGSLE

[0075] In some embodiments, the CDR sequences are SEQ ID NOs: 1-3; SEQ ID NOs: 1 , 4, and 5; SEQ ID NOs: 1 , 6, and 7; SEQ ID NOs: 1 , 8, and 9; SEQ ID NOs: 1 , 10, and 11 ; SEQ ID NOs: 1 , 12, and 13; SEQ ID NOs: 1 , 14, and 15; SEQ ID NOs: 16-18; SEQ ID NOs: 1 , 19, and 20; SEQ ID NOs: 21-23; SEQ ID NOs: 24-26; SEQ ID NOs: 1 and 65-66; and / or SEQ ID NOs: 27-29. In some embodiments, the CDR sequences are SEQ ID NOs: 1-3; SEQ ID NOs: 1 , 4, and 5; SEQ ID NOs: 1 , 6, and 7; SEQ ID NOs: 1 , 8, and 9; SEQ ID NOs: 1 , 10, and 11 ; SEQ ID NOs: 1 , 12, and 13; SEQ ID NOs: 1 , 14, and 15; SEQ ID NOs: 16-18; SEQ ID NOs: 1 , 19, and 20; SEQ ID NOs: 21-23; SEQ ID NOs: 24-26; and / or SEQ ID NOs: 27-29.

[0076] In some embodiments, the CDR sequences are SEQ ID NOs:1 and 10-11.

[0077] In some embodiments, the CDR sequences are SEQ ID NOs: 1 and 12-13.

[0078] In some embodiments, the CDR sequences are SEQ ID NOs: 16-18.

[0079] In some embodiments, the CDR sequences are SEQ ID NOs: 21-23.

[0080] In some embodiments, the immunoglobulin further comprises a signal peptide. In some embodiments, the signal peptide is pelB signal peptide.

[0081] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence set forth in Table 5; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequences set forth in Table 5, wherein the CDR sequences are as set forth in Table 5; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in Table 5.

[0082] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 30; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 30, wherein the CDR sequences are SEQ ID NOs: 1-3; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 1-3.

[0083] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 32; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 32, wherein the CDR sequences are SEQ ID NOs: 1 , and 6-7; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 6-7.

[0084] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 34; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 34, wherein the CDR sequences are SEQ ID NOs: 1 and 10-11 ; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 10- 11.

[0085] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 35; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 35, wherein the CDR sequences are SEQ ID NOs: 1 and 12-13; or iii) a conservativelysubstituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 12- 13.

[0086] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 37; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 37, wherein the CDR sequences are SEQ ID NOs: 16-18; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:16-18.

[0087] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 33; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 33, wherein the CDR sequences are SEQ ID NOs: 1 and 8-9; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 8-9.

[0088] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 39; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 39, wherein the CDR sequences are SEQ ID NOs: 21-23; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 21-23.

[0089] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 31; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 31, wherein the CDR sequences are SEQ ID NOs: 1 and 4-5; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 4-5.

[0090] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 36; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 36, wherein the CDR sequences are SEQ ID NOs: 1 and 14-15; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 14- 15.

[0091] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 38; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 38, wherein the CDR sequences are SEQ ID NOs: 1 and 19-20; or iii) a conservativelysubstituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 19- 20.

[0092] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 4031 ; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 40, wherein the CDR sequences are SEQ ID NOs: 24-26; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 24-26.

[0093] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 41 ; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 41, wherein the CDR sequences are SEQ ID NOs: 27-29; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 27-29.

[0094] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 56; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 56, wherein the CDR sequences are SEQ ID NOs: 59-61 ; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 59-61.

[0095] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 57; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 57, wherein the CDR sequences are SEQ ID NOs: 62-64; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 62-64.

[0096] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 58; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 58, wherein the CDR sequences are SEQ ID NOs: 1 , 65 and 66; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 1 , 65, and 66.

[0097] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 81 ; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 81 , wherein the CDR sequences are SEQ ID NOs: 82-84; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 82-84.

[0098] In some embodiments, the heavy chain variable region comprises at least residues Leu29, Ser30, Asn32, Thr33, Tyr37, Leu47, lle50, Asn52, Ser53, Gly54, Asp55, Arg56, Asn73, Asp98, Ser99, ProlOO, Leu101 , Pro103, Gly106, and Arg107 of SEQ ID NO: 40.

[0099] Another aspect of the disclosure includes an immunoglobulin that binds an IZLIMO1 protein, the immunoglobulin comprising a plurality of heavy chain variable regions each comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3 and optionally a signal peptide, wherein the amino acid sequences of said CDRs are:CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 2); andWVSTINNDGSIANCDR-H3 (SEQ ID NO: 3);TIGIKNTWRPCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 4); andWVSAITSGGGSTTCDR-H3 (SEQ ID NO: 5);APGTYHSGPIPD c)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 6); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 7);APGESPAFDGLVEVMD d)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 8); andWVSTINSGGGSTSCDR-H3 (SEQ ID NO: 9); orAPESATSAQAMANIPDCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 10); andWVSAINSDGGSTSCDR-H3 (SEQ ID NO: 11);APGRDSDYDIIPD f)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 12); andWVSIINSGGASTYCDR-H3 (SEQ ID NO: 13);APEGNPGPIATIPD g)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 14); andWVSTINSGGGKTSCDR-H3 (SEQ ID NO: 15);EPGYQRRYEYD h)CDR-H1 (SEQ ID NO: 16);SSYVMGCDR-H2 (SEQ ID NO: 17); andWVSTITSDGTSARCDR-H3 (SEQ ID NO: 18);APEEKIPIASIPE i)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 19); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 20);APGDSREHGSTWYAI PD j)CDR-H1 (SEQ ID NO: 21);SDYAMTCDR-H2 (SEQ ID NO: 22); andWVSSISSGGGSTRCDR-H3 (SEQ ID NO: 23).DIGWTFNNL k)CDR-H1 (SEQ ID NO: 24);SINTMGCDR-H2 (SEQ ID NO: 25); andLVAIINSGDRTNCDR-H3 (SEQ ID NO: 26);NVDSPLYPGRG l)CDR-H1 (SEQ ID NO: 27);SINVMGCDR-H2 (SEQ ID NO: 28); andLVAEITSGDTTNCDR-H3 (SEQ ID NO: 29)NIDSPRYGP m)CDR-H1 (SEQ ID NO: 59);SISAMTCDR-H2 (SEQ ID NO: 60); andWVARISSGGTTHCDR-H3 (SEQ ID NO: 61);NGIALTEANGSRRYNA n)CDR-H1 (SEQ ID NO: 62);SVNGMGCDR-H2 (SEQ ID NO: 63); andLVARITSGSYTDCDR-H3 (SEQ ID NO: 64);WYTDYVDNDGVQ o)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 65); andWVSTINNGGGSTSCDR-H3 (SEQ ID NO: 66); and / orAPGDYSDYVLQPDFGP)CDR-H1 (SEQ ID NO: 82);SVNGMGCDR-H2 (SEQ ID NO: 83); andMVARMTTGGCDR-H3YYLDFVGGEGGSLE(SEQ ID N0: 84)'

[0100] In some embodiments, the plurality comprises at least 2, at least 3, or at least 4, at least 5, at least 6 at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, or at least 24 heavy chain variable regions. In some embodiments, the plurality comprises at least 2 heavy chain variable regions.

[0101] In some embodiments, the immunoglobulin comprises two heavy chain variable regions. Any of the immunoglobulins described herein can be linked or fused as one construct. In some embodiments, the immunoglobulin is a bi-specific construct. In some embodiments, the immunoglobulin is a bi-paratopic construct.

[0102] In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of nanobodies from epitope binning group A (e.g., ZNb1 , ZNb2, ZNb3, ZNb5, ZNb6, ZNb7, ZNb8, or ZNb9) and the heavy chain variable region of nanobodies from epitope binning group D (ZNb15). In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of nanobodies from epitope binning group A (ZNb1 , ZNb2, ZNb3, ZNb5, ZNb6, ZNb7, ZNb8, or ZNb9) and the heavy chain variable region of nanobodies from epitope binning group E (ZNb13). In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of nanobodies from epitope binning group B (ZNb4 orZNblO) and the heavy chain variable region of nanobodies from epitope binning group D (ZNb15). In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of nanobodies from epitope binning group B (ZNb4 or ZNb10) and the heavy chain variable region of nanobodies from epitope binning group E (ZNb13). In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of nanobodies from epitope binning group D (ZNb15) and the heavy chain variable region of nanobodies from epitope binning group F (ZNb11 or ZNb12). In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of nanobodies from epitope binning group B (ZNb4 or ZNblO) and the heavy chain variable region of nanobodies from epitope binning group F (ZNb11 or ZNb12). In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of nanobodies from epitope binning group H (ZNb16) and the heavy chain variable region of nanobodies from epitope binning group D (ZNb15).

[0103] In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of e.g., ZNb6 and the heavy chain variable region of ZNb15. In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of ZNb6 and the heavy chain variable region of ZNb13. In some embodiments, the bi-paratopic construct comprises ZNb4 and ZNb15. In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of ZNb4 and the heavy chain variable region of ZNb13. In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of ZNb15 and the heavy chain variable region of ZNb11. In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of ZNb4 and the heavy chain variable region of ZNb11. In some embodiments, the bi-paratopic construct comprises the heavy chain variable region of ZNb16 and the heavy chain variable region of ZNb15.

[0104] In some embodiments, the immunoglobulin a multi-specific construct. In some embodiments, the immunoglobulin is a multi-valent construct.

[0105] In some embodiments, the plurality of heavy chain variable regions are fused or linked together. In some embodiments, the plurality of heavy chain variable regions are linked using a linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises multiple repeats of amino acid residues G and optionally S. In some embodiments, the linker comprises multiple repeats of amino acid sequence GGGS (SEQ ID NO: 87). In some embodiments, the linker comprises multiple repeats of the amino acid sequence GSGSGS (SEQ ID NO: 88). In some embodiments, the linker comprises multiple repeats of amino acid sequence GGGGS (SEQ ID NO: 89). In some embodiments, the linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 85). In some embodiments, the immunoglobulin further comprises one or more linker. In some embodiments, the linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 86). Examples of linkers can be found for example in e.g., Reddy Chichili, Vishnu Priyanka et al. “Linkers in the structural biology of protein-protein interactions.” Protein science : a publication of the Protein Society vol. 22,2 (2013): 153-67.

[0106] In some embodiments, the immunoglobulin is a nanobody. Nanobodies are approximately 10 times smaller than conventional antibodies (e.g., 12-15 kDa vs. 150 kDa).

[0107] In some embodiments, the immunoglobulin further comprises further domain(s) and / or moieties in addition to heavy chain variable region. In some embodiments, the further domain is a Fc domain. The Fc domain can be any species including a camelid species or human Fc domain. In some embodiments, the FC domain is a human Fc domain. In some embodiments,the domain is an albumin binding domain. In some embodiments, the immunoglobulin is fused to an antibody or binding fragment or scaffold (such as an IgM scaffold). In some embodiments, the antibody is an anti-albumin antibody or binding fragment.

[0108] In some embodiments, the immunoglobulin has a binding affinity for the IZLIMO1 protein measured by Kd and the Kd is a value of lower than about 100nM, optionally about 87 nM, when measured in a biolayer interferometry assay.

[0109] In some embodiments, the immunoglobulin has a binding affinity for the IZLIMO1 protein measured by Kd and the Kd is a value of lower than about 25nM.

[0110] In some embodiments, the immunoglobulin has a binding affinity for the IZLIMO1 protein measured by Kd and the Kd is a value of lower than about 87 nM, when measured in a biolayer interferometry assay.

[0111] In some embodiments, the immunoglobulin has a stronger binding affinity for the IZLIMO1 protein as compared to affinity of IZLIMO1 and JUNO, as measured by biolayer interferometry assay.

[0112] In some embodiments, the binding affinity is about as set forth in Table 4A, when measured in a biolayer interferometry assay.

[0113] The immunoglobulins described herein can inhibit binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 80% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 81% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 82% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 83% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 84% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 85% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 86% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 87% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 88% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about89% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZLIMO1 protein to oocytes and / or JUNO with at least about 90% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 91% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 92% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 93% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 94% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 95% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 96% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 97% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 98% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with at least about 99% inhibition. In some embodiments, the immunoglobulin inhibits binding of the IZUMO1 protein to oocytes and / or JUNO with 100% inhibition.

[0114] In some embodiments, the immunoglobulin is stable at room temperature.

[0115] Another aspect of the disclosure is an immunoglobulin which competes with the immunoglobulin described herein binding to an IZUMO1 protein.

[0116] In some embodiments, the immunoglobulin has at least 95% purity or has been purified to at least 95%.

[0117] Another aspect of the disclosure includes a nucleic acid molecule encoding any immunoglobulin described herein.

[0118] Another aspect of the disclosure includes a vector comprising any nucleic acid molecule described herein.

[0119] Another aspect of the disclosure includes a cell comprising any nucleic acid molecule described herein, any vector described herein, or expressing the immunoglobulin described herein.

[0120] Another aspect of the disclosure includes a composition comprising any the immunoglobulin described herein, any nucleic acid molecule described herein, any vector described herein, or any cell described herein, and a diluent or pharmaceutically acceptablecarrier. Due to the high solubility and stability of the immunoglobulins described herein, they can be developed as an injectables for systemic delivery, and / or intravaginal local drug delivery via a dissolvable vaginal film, intrauterine device, ring or gel.

[0121] In some embodiments, the composition comprises any immunoglobulin described herein and a diluent or pharmaceutically acceptable carrier or diluent.

[0122] In some embodiments, the composition is a lyophilized composition.

[0123] In some embodiments, the composition is a contraceptive composition.

[0124] In some embodiments, the composition comprises one or more nanoparticle.

[0125] In some embodiments, the composition is formulated is formulated for intravaginal administration. In some embodiments, the composition is formulated as a film, intrauterine device, ring or gel.

[0126] Another aspect of the disclosure includes a method of inhibiting binding of a JUNO protein with an IZUMO1 protein, the method comprising contacting the IZUMO1 protein with any immunoglobulin described herein. JUNO protein is found for example on oocytes and IZUMO1 is found for example on sperm. Accordingly, contacting IZUMO1 protein, includes for example contacting sperm that express IZUMO1 protein.

[0127] In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 80% inhibition of the binding of the JUNO protein (e.g. present on oocytes) with the IZUMO1 protein (e.g. present on sperm) . In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 81% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 82% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 83% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 84% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 85% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount ofthe immunoglobulin sufficient to provide at least about 86% inhibition of the binding of the JUNO protein with the IZUM01 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 87% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 88% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 89% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 90% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 91% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 92% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 93% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 94% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 95% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 96% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 97% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 98% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the method comprises contacting the IZUMO1 protein with an amount of the immunoglobulin sufficient to provide at least about 99% inhibition of the binding of the JUNO protein with the IZUMO1 protein. In some embodiments, the methodcomprises contacting the IZLIMO1 protein with an amount of the immunoglobulin sufficient to provide 100% inhibition of the binding of the JUNO protein with the IZUMO1 protein.

[0128] Another aspect of the disclosure includes a method of inhibiting fertilization, the method comprising contacting IZUMO1 protein with an immunoglobulin described herein.

[0129] In some embodiments, the IZUMO1 protein contacted is expressed on spermatozoa (e.g. on sperm), the spermatozoa optionally in a subject.

[0130] In some embodiments, the subject is a human.

[0131] In some embodiments, the contacting comprises administering the immunoglobulin described herein or the composition described herein, to the subject, , optionally intravaginally or systemically, for example prior to intercourse. For example, the method may comprise the immunoglobulin described herein being present in a composition and the composition being administered to / in a vaginal canal, thereby allowing contact between IZUMO1 protein and the immunoglobulin described herein.

[0132] In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 80%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 81%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 82%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 83%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 84%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 85%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 86%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 87%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 88%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 89%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 90%. In some embodiments, theimmunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 91%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 92%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 93%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 94%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 95%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 96%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 97%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 98%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 99%. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by 100%.

[0133] Another aspect of the disclosure includes use of the immunoglobulin described herein or any composition described herein as a contraceptive or in the manufacture of a contraceptive.

[0134] In some embodiments, the cell is a yeast cell (e.g., P. pastoris or S. Cerivisiae). In an embodiment, the cell is a CHO cell. In some embodiments, cell is a ExpiCHO cell. In some embodiments, the cell is an Expi293 cell. In some embodiments, the cell is an E. coli cell.

[0135] In some embodiments, the contraceptive is a human contraceptive.

[0136] Another aspect of the disclosure includes a protein complex comprising an IZLIMO1 protein interacting with any immunoglobulin described herein. The IZLIMO1 protein in the protein complex, can be a fragment of IZLIMO1 protein.

[0137] An aspect of the disclosure includes a method of making an immunoglobulin described herein. In some embodiments, the method of making comprises one or more method described herein (e.g., in the Examples). In some embodiments, the method comprising administrating an IZLIMO1 protein to a subject, optionally a llama, isolating the immunoglobulin and / or cells expressing the immunoglobulin specific or selective for the IZLIMO1 protein.

[0138] Another aspect of the disclosure includes a method of making any immunoglobulin described herein comprising: a) introducing any nucleic acid described herein or any vector described herein, in a suitable cell or b) culturing any cell described herein; and isolating the immunoglobulin.

[0139] Another aspect of the disclosure includes a kit or immunological assay comprising one or more of the immunoglobulin described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein, and optionally a container or vial. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit comprises a plurality of the immunoglobulin described herein. In some embodiments, the kit further comprises a delivery vehicle e.g., a film, gel, or intrauterine device. In some embodiments, the kit further comprises one or more nanoparticle.

[0140] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0141] The following non-limiting examples are illustrative of the present disclosure:EXAMPLESExample 1MethodsAntigen production

[0142] The genes corresponding to full-length human IZUMO1 (GenBank accession number: NM_182575, residues 1-350) was codon optimized for expression in Drosophila melanogaster and gene synthesized (Integrated DNA Technologies). The DNA sequence encoding the extracellular regions of IZUMO1 (residues 22-254) with a BiP signal peptide was subcloned into a modified pMT-puromycin expression vector. Protein constructs contain a thrombin cleavage site and 10X-His affinity tag at the C-terminus. The resulting wild-type IZUMO122-254 pMT expression plasmid was stably transfected in Drosophila S2 cells (Invitrogen) using Effectene transfection reagent (Qiagen), according to manufacturer’s protocol.

[0143] Briefly, Drosophila S2 cells were cultured in Schneider’s medium (Lonza) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) plus 1X antibiotic- antimycotic (Gibco), and propagated at 27°C. The day before transfection, 3x106cells were seeded per well in a 6-well plate (Corning) with 3.0 ml complete growth medium and incubated overnight. On the day of transfection, 2 pg expression plasmid was mixed with the transfection reagents and the transfection complexes were added dropwise onto the S2 cells. At 72 hours post-transfection, the cultured media were replaced with fresh S2 growth media supplemented with 6 pg ml’1puromycin (Bioshop). Subsequently, S2 cells were gradually adapted to FBS-free Insect-XPRESS growth media (Lonza) with 6 pg ml’1puromycin. Stably transfected cells were grown to 1x107cells ml’1in Insect-XPRESS growth media using vented 2 L polycarbonate Erlenmeyer flasks (VWR) at 27°C. Protein expression was induced with 500 pM final concentration of sterile-filtered CuSC Cultured media were harvested 6-days post-induction, clarified by centrifugation at 6750xg for 20 minutes, concentrated and buffer exchanged into Ni- NTA binding buffer (20 mM Tris-HCI [pH 8.0], 300 mM NaCI, 20 mM imidazole) using a Centramate tangential flow filtration system (Pall Corp.) IZUMO1 protein was purified by Ni-NTA metal affinity chromatography. Eluted samples were thrombin (EMD Millipore) digested at 22°C for 24 hours (1 :2000 (w / w) enzyme to protein ratio) while being dialyzed against 1X HBS. IZUMO1 was purified by size exclusion chromatography on a Superdex-75 Increase 10 / 300 column equilibrated with HBS. Peak fractions were pooled and protein concentrations were quantified by measuring A280.Immunization

[0144] Three llamas were sourced and held under quarantine for 2 weeks at VIB Nanobody Core (Brussels, Belgium). During this period, the animals were screened for (infectious) diseases & monitored for stress behavior. After approval by the authorized veterinarian, the animals were subject to immunization. Immunizations and handling of the animals were performed according to directive 2010 / 63 / EU of the European parliament for the protection of animals used for scientific purposes and approved by local Ethical Committee for Animal Experiments.

[0145] Each animal was injected subcutaneously for 6 times on a weekly basis, each time with an emulsion of adjuvant and purified recombinant IZUMO1 protein (about 100 pg per injection per animal). The adjuvant used was Gerbu P. The general health status (eating habit, body temperature, etc.) of the animals was monitored daily throughout the immunization period. The animals remained healthy during the whole procedure.Construction of independent VHH libraries

[0146] Three phage display VHH libraries, one per vaccinated animal, were constructed from the llamas’ lymphocytes to screen for the presence of antigen specific nanobodies. Four & 8 days after last injection (4 d.p.i. & 8 d.p.i., respectively), each time about 100 ml anticoagulated blood was collected for the preparation of peripheral blood lymphocytes (PBLs). Total RNA was prepared from PBLs from each blood sample separately (4 d.p.i. & 8 d.p.i.). For each animal, about 50 pg of pool of the total RNA from 4 d.p.i. and 8 d.p.i. PBLs (25 pg from each blood sample) was used as template for first strand cDNA synthesis with oligodT primer. Using the cDNA samples obtained from PBL-derived RNAs, the VHH encoding sequences were amplified by PCR, digested with Pstl and Notl restriction enzymes, and cloned into the Pstl / Notl sites of the phagemid vector pMECS. Electro-competent E. coli TG1 cells were transformed with the recombinant pMECS vector, resulting in VHH (Nanobody) libraries. For each library, the percentage of the colonies harboring the vector with the right insert size was estimated by the PCR analysis of randomly picked colonies. In pMECS vectors, the Nanobody sequence is followed by a linker (e.g., AAA), HA tag (e.g., YPYDVPDYGS (SEQ ID NO: 67)) and His6tag (e.g., HHHHHH (SEQ ID NO: 68)).Isolation of nanobodies specific against IZUMO1

[0147] Each of the 3 immune libraries, derived from the animals immunized with human IZUMO1 , was separately panned on solid-phase coated human IZUMO1 (100 pg / ml in 100 mM NaHCO3 pH 8.2) for 3 rounds. Here also, the enrichment for antigen-specific phages was assessed after each round of panning by comparing the number of phagemid particles eluted from antigen-coated wells with the number of phagemid particles eluted from negative control (uncoated blocked) wells. Subsequently, for each library, 190 colonies from round 2 and round 3 (95 from each round) were randomly selected and analyzed by ELISA for the presence of antigenspecific Nanobodies in their periplasmic extracts (ELISA using crude periplasmic extracts including soluble Nanobodies). The antigen used for panning and ELISA screening was the same as the one used for immunization. Uncoated blocked wells served as negative control.

[0148] In summary, the panning and ELISA screening of 3 different immune VHH libraries resulted in the identification of 235 different human IZUMO1 -specific Nanobodies. These 235 unique Nanobodies belong to 48 different B-cell lineages (CDR3 groups).Generation of periplasmic extracts (PE)

[0149] The nanobody clones were transformed and inoculated onto LB-agar ampicillin plates. The clones were then seeded in 1 ml of TB + ampicillin medium. After 6h, nanobody production was induced with final concentration of 1.2 mM IPTG. After overnight production, the cultures were spun down and cells were resuspended in TES buffer (Tris + sucrose & EDTA). Two hours later, H2O was added & the suspension was left for another 4h. The bacteria were removed through centrifugation, after which the PEs were used for the off-rate experiments or frozen at -80°C.Off-rate determination

[0150] In order to rank the nanobody binders, off-rate determination was performed using biolayer interferometry (BLI). The biotinylated human IZLIMO1 was used at 5 pg / ml in the “Ligand scouting immobilization conditions” protocol for streptavidin (SA)-biosensors as detailed in ForteBio Technical Note 26, in conjunction with a ForteBio Octet Red machine. Per Nb, 200 pl of PE was mixed with 2 pl of 10% (v / v) Tween20-PBS in a well of a 96 well black-plate to reduce aspecific interactions. For each set of experiments, PE from an E. coli containing the empty pMECS vector was used as negative control. This plate was loaded into a ForteBio Octet Red and brought into contact with antigen-coated Octet tips. Consequently, the binding profile for each clone was determined. Using the ForteBio Data Analysis Software, the blanks were subtracted and the curves were aligned. Based on these curves, the off-rates were calculated using a 1 :1 binding model.Interference assay of anti-IZUMO1 nanobodies

[0151] In order to determine whether the anti-IZUMO1 nanobody binders inhibited the human JUNO-IZUMO1 interaction, a BLI-based interference assay was performed. Biotinylated human IZUMO1 was coated on Octet SA tips, then the coated tips were allowed to interact with PEs containing anti-IZUMO1 Nbs for 600 seconds, till the saturation was reached. During the next step, tips were soaked in a solution containing human JUNO at 200 nM together with the PEs containing exactly the same Nbs, to avoid leaving the epitope exposed. The binding of JUNO on coated IZUMO1 , previously bound with Nbs, was detected, in order to check the capacity of these Nbs to inhibit the JUNO / IZUMO1 interaction. Tips without Nbs or with only JUNO at 200 nM were used as references.Orthogonal fluorescence-based screening assay

[0152] As an orthogonal assay to the BLI-interference assay, a second interference assay based on fluorescence was developed. The batch of 46 semi-purified anti-IZUMO1 nanobodiesfrom PE were diluted to a stock of 100 pg / mL in PBS buffer. Tag-removed recombinant IZUMOI22- 254 was biotinylated in a 1 :1 (biotin: protein) molar ratio (EZ-link sulfo-NHS-LC-biotin; Thermo Scientific, cat#A39257) and tag-removed recombinant JUN02o-228was labeled with fluorescein in a 1 :1 (dye:protein) molar ratio (NHS-Fluorescein; Thermo Scientific, cat#46410), both according to manufacturer’s instructions. The excess biotin and dye were removed via overnight dialysis into PBS buffer. A streptavidin-coated 96-well plate (Thermo Scientific, cat#15119) was first washed with 200 pL of assay buffer (PBS, 0.1 % (w / v) BSA, 0.05% (v / v) Tween-20). Then, 100 pL of Biotin-IZUMOI (10 pg / mL), diluted in assay buffer, was immobilized onto the wells by incubating at room temperature (24 °C) for 2 hours. The wells were washed with 200 pL of assay buffer three times and was further blocked using 200 pL of assay buffer for 2 hours (24 °C). 100 pL of anti-IZUMO1 nanobodies at final concentration of 50 pg / mL, 12.5 pg / mL, or 1 pg / mL were diluted from the stock in assay buffer, added to the wells, and incubated for 45 minutes (24 °C). After another three washes, 100 pL of fluorescein-JUNO were added to the wells and incubated for 45 minutes (24 °C). The solution was discarded, and the wells were washed 3-times, before a final 100 pL of PBS buffer was added and the end-point fluorescence emission intensity was detected using the Synergy neo2 multi-mode plate reader (BioTek; excitation = 487 nm, emission = 528 nm). A blank control with only immobilized biotin- IZLIMO1 and the buffer added in place of nanobodies and fluorescein-JUNO and a negative control with the only the addition of biotin- IZUMO1 bait and fluorescein-JUNO, but without any nanobodies, were both included. The Relative Fluorescence Unit (RFU) obtained from the blank control well was subtracted from the RFU of the experimental wells and the blanked values were compared to the RFU of the negative control (blank-corrected). The degree of Inhibition for the nanobodies was calculated using the formula:Inhibition

[0153] Results from both the Octet biolayer interferometry and the fluorescence-based assay were used to rank the anti-IZUMO1 nanobodies.Epitope binning assay

[0154] In order to determine whether the anti-IZUMO1 nanobodies bound to the same epitope, a BLI-based epitope binning assay was performed. The target antigen was coated on a series of Octet SA tips. Each series of tips was first soaked in a periplasmic extract containing one specific Nb (termed first Nb). After the response signals reached saturation (where possible with extracts), the tips were transferred to a mix of PEs containing again the first Nb (at the sameconcentration) plus a different Nb (termed secondary Nb) for each tip of the series. Secondary Nbs that still bind the target antigen in the presence of the first Nb will show an increased response, as compared to the binding response of the first Nb alone. As a control, one tip was also incubated with the first Nb in the second incubation step, which should not lead to an increased response. This was repeated for each Nb as primary Nb until all Nbs were tested against each other in both directions (as primary and as secondary binder). From the response data, the bins were deduced for each Nb.Anti-IZUMO1 Nanobodies Expression and Purification

[0155] Twelve Anti-IZUMO1 Nanobodies sequences were cloned into pET22b vector with pelB as a signal peptide to guide for periplasmic localization, thrombin cleavage site and a His- Tag made of 10-histidine residues for affinity chromatography purpose (Fig. 2). A selection gene, ampicillin was included to provide resistance in ampicillin-containing media. Every cloned Anti- IZLIMO1 nanobody was transformed in E. coli (DE3) and plated onto LB-agar. A single colony was inoculated into a pre-culture of ampicillin-containing LB-media. 10 mL of the pre-culture was inoculated into 1 L of Terrific Broth (TB) media (supplemented with 100 pg / mL Ampicillin and 0.1 % glucose), which was then incubated at 37°C with a shaking rate of 180 rpm until the density reached an OD600 between 0.6 and 0.9. Once the OD600 was reached, 1 mM of isopropyl-b-D- thiogalactopyranoside (IPTG) was added to induce the protein expression. Then, further incubated for 4 hours at 37°C. The media was centrifuged at 5000 rpm for 30 minutes and the cell pellets were collected. To extract the periplasmic content, an osmotic shock was conducted, the pellets were thawn at room temperature and resuspended with 12 mL TES buffer (0.2 M Tris pH 8.0, 0.5 mM EDTA, 0.5 M sucrose). Shook for 1 hour at 4°C, then 24 mL of ! dilution of TES was added and further incubated at 4°C for additional 2 hours while shaking. The extracts were centrifuged for 30 minutes at 4°C at 10,000 rpm and the supernatant (periplasmic extracts) was collected. The nanobodies were all purified using Immobilized Metal Affinity Chromatography (IMAC) with 0.4 mL of Ni-NTA charged Magnetic beads (Genscript, 25% slurry) per extract from 1 L of grown media. Then, nutated for 1 hour at room temperature. The C-terminus 10xHis of the nanobody chelate Ni(ll) resulting in its attachment into Ni-NTA Magnetic Beads. The beads were washed with the buffer A (20 mM Tris pH 7.5, 300 mM NaCI and 20 mM imidazole) and the nanobody was eluted with buffer B (20 mM Tris pH 7.5, 300 mM NaCI and 500 mM imidazole).

[0156] To remove the 10xHis-Tag, thrombin digestion was performed (1 Unit of thrombin I mg of nanobody) using SnakeSkin Dialysis Tubing 10 K MWCO overnight in PBS (1x) pH 7.4. Size exclusion chromatography (SEC) (AKTA Purifier, with Superdex 75 Increase) using PBS (1x)pH 7.4 as a buffer was conducted to further purify the nanobody. The fractions of interest were collected and concentrated using Amicon (3kD MWCO, 4mL). The concentration of the nanobody was determined by Nanodrop protein A280, then aliquoted and stored at -80°C.Quality Controls

[0157] Quality control analysis of the nanobody was performed including Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophpseris (SDS-PAGE) and mass spectrometry with Matrix Assisted Laser Desorption / lonization Time-of-Flight (MALDI-TOF) (Figs. 3A-B and 4). For MALDI- TOF, a sandwich sample spotting was used, 0.6 pM of Sinapinic acid (prepared by dissolving 20 mg in 50% acetonitrile) was spotted onto the target plate and air-dried, 0.6 pM of nanobody sample at a concentration of 1 mg / mL was added on the dried matrix and let to dry for cocrystallization. Finally, 0.6 pM of the matrix was placed on top. Once everything dried, the samples were assessed by MALDI-TOF mass spectrometer (Bruker). The nanobodies were best detected using Linear Positive (LP) mode and data were processed using FlexAnalysis.Measurement of Anti-IZUMO1 thermal stability

[0158] To characterize thermal unfolding of the Anti-IZUMO1 nanobodies, nanoTEMPER Tycho NT.6 was used. Purified nanobodies in PBS (1x) pH 7.4 were loaded onto the capillary tubes and placed in the system, which performs varying temperature from 35 to 95°C and provides the inflection temperature (Ti) of the unfolding transition in the 350 nm I 330 nm ratio signal.

[0159] Table 1 : Summary of the expression yield of Anti-IZUMO1 nanobodies with and without 10xHisMeasurement of the binding affinities of Anti-IZUM01 nanobodies

[0160] Biolayer Interferometry (BLI) was used to measure the binding affinity of the 12 Anti-IZUMO1 Nanobodies to IZLIMO1. The measurement was conducted using a single-channel BLItz instrument (ForteBio). The 12 Anti-IZUMO1 Nanobodies were expressed using pET-22 b vector in E. coli (DE3), purified with Immobilized Metal Affinity (IMAC) chromatography and stored in PBS (1x) pH 7.4. Unlike, Nanobodies, IZUMO1 was expressed using pTM-puro expression plasmid in Drosophila S2 cells (Invitrogen), purified with Ni-NTA chromatography and stored in 50 mM HEPES pH 7.5, 150 mM NaCI with 5% Glycerol. Prior to biotinylation, IZUMO1 was further purified using size chromatography with PBS (1x) pH 7.4 as a buffer to get rid of glycerol.

[0161] EZ-Link sulfo-NHS -LC-biotinylation (Thermo Pierce) was mixed with IZUMO1 in 1 :1 Molar ratio. The mixture was incubated for 2 hours on ice. To remove any excess of biotin, the mixture was dialyzed overnight in PBS (1x) pH 7.4 with SnakeSkinTM Dialysis Tubing 10 K MWCO. The biosensors coated with streptavidin (SA) were hydrated for 10 minutes in BLI rehydration buffer (PBS (1x) pH 7.4, 0.5 mg / mL BSA and 0.01% (v / v) Tween-20). A stock solution of 10 pM of biotinylated IZUMO1 (bait) was prepared in BLI kinetics buffer (PBS (1x) pH 7.4, 0.1 mg / mL BSA and 0.01% (v / v) Tween-20). This was further diluted with the BLI kinetics buffer to afford a working concentration of 200 nM and immobilized onto SA-coated biosensor for 120s. Different concentrations of each Anti-IZUMO1 nanobody (analyte) were prepared from 10 pM stock solution in BLI kinetics buffer. The analyte association to biotinylated IZUMO1 (bait) was measured over 120s at 22°C. The dissociation of the analyte was also measured over 120s by immersing the SA biosensor into BLI kinetics buffer. To detect non-specific binding, the negativecontrols were performed: BSA and BLI kinetics buffer alone against biotinylated IZLIMO1 (baity- loaded SA biosensors. All the experiments were conducted in triplicate. The data were analyzed and fitted globally to a 1 :1 binding model. The equilibrium dissociation constant (KD) as well as association (kaor kon) and dissociation (fa) rate constants were obtained. The results are expressed as a mean ± standard error of the mean.Quantification of the inhibitory effect of Anti-IZUMO1 nanobodies on IZUMO1-Juno interaction

[0162] Biolayer Interferometry (BLI) was also used for the quantification of the inhibitory effect of Anti-IZUMO1 nanobodies on the interaction between IZLIMO1 and JUNO. An eightchannel Octet ForteBio instrument was used. The biotinylation of IZUMO1 (bait) was conducted as mentioned for the binding affinity measurement. Streptavidin-coated (SA) biosensors were hydrated in BLI rehydration buffer (PBS (1x) pH 7.4, 0.5 mg / mL BSA and 0.01% (v / v) Tween-20) for 10 minutes. The SA biosensors were equilibrated in BLI kinetics buffer (BPS (1x) pH 7.4, 0.1 mg / mL BSAand 0.01% (v / v) Tween-20) for 60 seconds. 200 nM of the biotinylated IZUMO1 (bait) was then loaded onto the SA biosensors for 600 seconds then washed in the BLI kinetics buffer for 60 seconds to get rid of unbound bait molecules. For every anti-IZUMO1 nanobody (analyte), varying concentrations (0 nM to 300 nM) were prepared from 10 pM stock solution in BLI kinetics buffer and bound to IZUMO1 for 600 seconds (associationl). Subsequently, the biosensors (SA- IZUMO1-Anti-IZUMO1) were immersed into a mixture of 500 nM JUNO prepared in BLI kinetics buffer and the analyte (anti-IZUMO1 from 0 to 300 nM) over 600 seconds to measure the association2.

[0163] The experiments were conducted in triplicate and the raw data made of associationl and association2 were processed. The association2 data was used to determine the half maximal inhibitory concentration (IC50). The dataset of the first 20 seconds of the association2 were considered to reflect the binding initial rate.% of inhibition = (1 - (wavelength shift of A / Wavelength shift of B)) x 100

[0164] A: The value of the wavelength shift of any given concentration. B: The value of the wavelength shift of 0 nM of nanobody with 500 nM of Juno.

[0165] All non-linear line fitting (a HillSlope of -1 .0 model) analyses were performed using GraphPad Prism (version 5.0, GraphPad Software Inc., San Diego, CA). Data were presented as mean ± standard error of the mean (SEM), n = 3.Hamster oocyte-human sperm in vitro fertilization assay

[0166] For anti-IZUMO1 nanobodies, an initial hamster oocyte-human sperm in vitro fertilization assay was used to determine the ability of nanobodies to block fertilization. Due to the scarcity of human oocytes, hamster oocyte / human sperm was used as a surrogate assay. Zona- free hamster oocytes were commercially purchased and used in an in vitro fertilization assay. Zona-free oocytes were inseminated overnight with -4000 capacitated motile human sperm in the presence of anti-IZUMO1 nanobodies at various concentrations. Removing the ZP allows polyspermia, thus the fertilization index (i.e. the number of sperm fused per oocyte) was measured as an indication of insemination interference. For analysis, oocytes were treated with the DNA- specific fluorophore Hoechst 33342 (Sigma-Aldrich) and observed using fluorescence light microscopy (Nikon Eclipse E600) and spinning disk confocal microscopy. Fusion is considered to have occurred when sperm nuclei are detected in oocytes.Human in vitro fertilization assay

[0167] Nanobodies with desirable characteristics (high affinity and nanomolar IC50 values) were tested using a zona-free human in vitro fertilization (IVF) assay. The human IVF is the most biologically relevant assay to test the nanobodies. This is the only test that can be performed in vitro with human samples, as cumulus-intact human IVF is prohibited in France by bioethics law. Human sperm and oocytes were obtained from consenting patients treated through an assisted reproductive technology program at the Cochin Hospital (Paris). Only human oocytes that could not be used for the patient’s fertility treatment were used for research in accordance with French laws. In v / tro- matured and unfertilized metaphase II oocytes were used for IVF. The ZP was removed chemically using acidic Tyrode’s solution (Sigma-Aldrich), and the resulting oocytes washed in culture medium prior to incubation at 37°C and 5% CO2 for recovery. Zona- free oocytes were inseminated overnight with -4000 capacitated motile human sperm in the presence of anti-IZUMO1 nanobodies at various concentrations. Removing the ZP allows polyspermia, thus the fertilization index (i.e. the number of sperm fused per oocyte) was measured as an indication of insemination interference. For analysis, oocytes were treated with the DNA- specific fluorophore Hoechst 33342 (Sigma-Aldrich) and observed using fluorescence light microscopy (Nikon Eclipse E600) and spinning disk confocal microscopy. Fusion is considered to have occurred when sperm nuclei are detected in oocytes. The in vitro zona-free fertilization assay was used in several previous studies (Jean et al. 2019 Hum Reprod 34, 118-26; Barbaux et al. 2020 Sci Rep 10:5335).RESULTS

[0168] Discovery of anti-IZUMO1 nanobodies: Llama immunizations at VIB (Brussels) resulted in the identification of 235 anti-IZUMO1 nanobodies, belonging to 48 unique CDR3 groups. The top 46 anti-IZUMO1 Nbs were arbitrarily selected for JUNO-IZUMO1 interference assay.

[0169] Table 2: Inhibition Assay Results

[0170] Based on the results of the BLI interference assay, 26 anti-IZUMO1 Nbs were identified with >80% binding inhibition, thus exceeding expectation.

[0171] The top 12 nanobodies belonging to 3 different epitope binning groups were selected based on their k-off rate and ability to block the JUNO-IZUMO1 interaction in a BLI assay (Table 3). All 12 nanobodies were sufficiently expressed in larger E. coli cell cultures and displayed Kd values in the nanomolar range (between 5.7-86.3 nM) (Tables 3, 4A). The majority of these nanobodies have Kd values below 25 nM. Nb11-25644 (Kd=5.7 nM), has one order of magnitude better in binding affinity than the natural affinity of IZUMO1-JUNO. Eight other nanobodies (Nb2-25607, Nb3- 25545, Nb4-25568, Nb6-25619, Nb7-25570, Nb9-25587, Nb10-25648, and Nb12-25740) have Kd values equal or better than the affinity of IZUMO1-JUNO. Strong IC50 values (4.0-39.6 nM) were observed for all 3 epitope binning groups in anti-IZUMO1 nanobodies (Table 3, 4A).

[0172] Table 3. kon, kotf, and Kd values for anti-IZUMO1 nanobodies

[0173] Downselection of nanobodies for lead optimization and profiling: Nanobodies were downselected based on displaying: nanomolar affinities and IC50 values, unique epitope binning groups, milligram quantity expression yields and >95% purity (no degradation products), >90% inhibition in hamster IVF assays.

[0174] 12 nanobodies displayed Kd values in the nanomolar range between 5.7-86.3 nM, with the majority having Kd values better than 25 nM (Table 4A; Figs. 6A-L). Interestingly, strong IC50 values (4.0-39.6 nM) were observed for all anti-IZUMO1 nanobodies (Table 4B; Fig. 7).

[0175] Using a surrogate hamster oocyte-human sperm based in vitro fertilization assay at Institute Cochin / INSERM (Paris), 7 nanobodies within epitope groups A and B incubated at 10 microgram / ml have >90% inhibition of fertilization (Nb3-25545, Nb6-25619, Nb10-25648, Nb1- 25600, Nb4-25568, Nb5-25557, Nb8-25553) (Fig. 1A). Results from human IVF assay demonstrate that Nb5, Nb, 8 and Nb10 and Nb6 inhibited fertilization, with Nb10 and Nb6 having >95% inhibition in human IVF assay.

[0176] To characterize thermal unfolding of the Anti-IZUMO1 nanobodies, inflection temperatures (Ti) of the nanobodies were obtained. #4 Anti-IZUMO1 had a Ti = 63.5 ± 0.96 °C, #5 Anti-IZUMO1 had a Ti = 62.1 ± 1.03 °C, #6 Anti-IZUMO1 had a Ti = 56.9 ± 0.85 °C, #8 Anti- IZUMO1 had a Ti = 62.9 ± 0.97 °C (Figs. 5A-D).

[0177] In summary, anti-IZUMO1 nanobodies that are effective in blocking in vitro fertilization were discovered (Figs. 1A-C). These nanobodies are selected as candidates for the development of non-hormonal contraceptives.

[0178] Table 4A: Summary of the KD values of the 12 Anti-IZUMO1 nanobodies

[0179] Table 4B: Summary of the IC50 values of the 12 Anti-IZUMO1 nanobodies

[0180] Table 5: Nanobody Sequences and CDRs (His tag in italics and thrombin cleavage site bolded).Example 2

[0181] Methods for manufacturing immunoglobulins using mammalian expression systems. Immunoglobulins such as nanobody manifold constructs (bivalent, biparatopic, Fc fusions and nanoparticles) are secreted in a mammalian expression system (Chinese hamster ovary cells or HEK293 cells), which is the standard in the production of therapeutic antibodies and nanobodies. Constructs are codon optimized and gene synthesized into the pcDNA3.4 vector (Invitrogen). IgK signal peptide sequence is inserted prior to the start of the nanobody gene to target the nanobodies for secretion. The expression vector is transfected into ExpiCHO or 293F or Expi293 cells and grown in a ExpiCHO / Expi293 / 293 expression medium at 37°C and 120 rpm. A time-course induction profile is performed to determine expression levels. Nanobodies are purified from the supernatant using Protein A chromatography followed by FPLC size exclusion chromatography (Superdex 200 Increase).Example 3Biophysical characterization

[0182] To understand the stability, aggregation potential and quality of the nanobodies, biophysical characterization can be performed.

[0183] Circular dichroism spectroscopy: Circular dichroism spectroscopy can be performed to understand the stability of the nanobodies. Nanobody samples can be dialyzed in 5 mM KH2PO4, 100 nM NaF pH 7.5 and diluted to 0.1 mg / mL or 0.2 mg / mL. CD wavelength scans of the nanobodies can be measured at 25 °C over the range of 185 to 250 nm in a 1 mm quartz cuvette (Helma) using a Jasco J- 1500 spectropolarimeter. CD spectra can be baseline-corrected, averaged over 8 accumulations and converted to mean residue ellipticity (0MRE).

[0184] To maximize the CD signals for the thermal melt experiments, the wavelength at which each nanobody displayed the greatest change in ellipticity upon thermal denaturation can be selected. Thermal denaturation assays can be conducted by increasing the temperature from 25 to 91 °C, with a full CD wavelength scan (190 to 260 nm) measured every 2 °C. All data can be baseline-subtracted and the change in ellipticity can be normalized between 0 (folded) and 1 (unfolded). The resultant curve can be fit with a non-linear sigmoidal function using GraphPad Prism 9 (GraphPad Software, San Diego, CA) to determine the melting temperature (Tm).

[0185] Differential static light scattering: DSLS can be used to determine the aggregation potential of the nanobodies. Nanobodies can be diluted to 1 mg / mL in HBS buffer and 10 pL of each nanobody was added to a 384-well black plate (Corning) and covered with 10 pL of mineral oil to prevent evaporation. A light scattering temperature scan from 20.0 to 93.2 °C (increasing 1.0 °C / min) at a fixed wavelength of 620 nm can be performed using the Stargazer2 DSLS instrument. The data can be processed and analyzed using the Stargazer AIR software to calculate the aggregation temperature (Tagg) for each nanobody.Example 4

[0186] Additional data (more replicates) was collected for the hamster oocyte / human sperm IVF assay to characterize the anti-IZUMO1 nanobodies (Figs. 1 B-1C). The numbers above the error bars refer to the number of replicates.

[0187] Table 6: Nanobody Epitope Binning

[0188] Table 7: Residues on ZNb11 that interact with IZUMO1 (based on crystal structure)Stress tests

[0189] We performed thermal and chemical stress tests on anti-IZUMO1 nanobodies and then monitored their effects on binding affinity, aggregation and protein recovery.

[0190] For thermal stress tests, anti-IZUMO1 nanobodies were incubated at -20C for 30 days, 4C for 30 days, 30C for 30 days or 60C for 24 hours. After stresses, anti-IZUMO1 nanobodies were measured for the binding to recombinant IZLIMO1 by BLI, their ability to aggregate (Tagg), and the fraction of anti-IZUMO1 nanobody that was lost due to the stress as compared to unstressed samples.

[0191] For chemical stress tests, anti-IZUMO1 nanobodies were incubated either at pH 3.7 or pH 9.0. After stresses, anti-IZUMO1 nanobodies were measured for the binding torecombinant IZLIMO1 by BLI, their ability to aggregate (Tagg), and the fraction of anti-IZUMO1 nanobody that was lost due to the stress as compared to unstressed samples.

[0192] Table 8A: Summary of Stress Test Results on IZUMO1 nanobodies: Binding affinity (Kd) to IZUMO1.

[0193] Table 8B: Summary of Stress Test Results on IZUMO1 nanobodies: Aggregation temperature (Tagg) of stressed nanobodies.

[0194] Table 8C: Summary of Stress Test Results on IZUMO1 nanobodies: Percent protein recovery after stress tests.ppt: Protein precipitated during stress test nd: Not determined tbd: To be determined agg: Protein aggregatedCrystallography of anti-IZUMO1-IZUMO1 complexes

[0195] In order to under how the anti-IZUMO1 nanobodies interact with IZLIMO1 target, we carried out X-ray crystallography. To date, we have one crystal co-complexed structured with ZNb11 and IZUMO1.

[0196] The structural features reveal that ZNb11 binds to IZLIMO1 in the same location as JUNO. Upon analyzing the interacting residues, it is observed that like JUNO, ZNb11 binds to IZUMO1 primarily through the hinge region. The 4HB and the Ig-like domain also participate in the interaction but to a lesser extent. When comparing the key interactions, we observed that ZNb11 has a 77% overlap with the JUNO binding site, thus allowing ZNb11 to directly compete and block JUNO interaction.

[0197] Table 9: Data Collection statistics for crystallization of ZNb11.Resolution34.05-2.98 (3.28- 2.98) rangeSpace group P 31 2 1 a=110.6 b=110.6 c=145.2Unit cell90, 90, 120Total512,935 (23,436} reflectionsUnique13,261 (663} reflectionsMultiplicity 38 7 (35 3}Completeness94 4 (84 9}(%}Mean8 7 (1 7} l / sigma(l }R-merge 0 588 (3 365}CCI / 2 0 997 (0 672}Crystals diffracted to 3 ABinding of multiple anti-IZUMO1 nanobodies

[0198] Combining multiple nanobodies that bind to different epitopes is expected to improve potency (e.g., inhibition of fertilization of the nanobodies). Here, a BLI-based assay was used to determine which anti-IZUMO1 nanobody is capable of being combined with another anti- IZLIMO1 nanobody to form a ternary complex. First, the first nanobody was bound to an immobilized IZLIMO1, then a second nanobody was allowed to associate. If binding was observed, this would suggest that the two nanobodies are not mutually exclusive and capable of binding to IZLIMO1 at the same time.

[0199] Table 10 below summarizes results related to the prospect of making ternary complexes with anti-IZUMO1 nanobodies from different binning groups.

[0200] Table 10: Ternary complexes summary table.Legend:Y= yes;N= No;Y* = yes but the order matters;? = Not tested;Grey colour = Crystallization chaperone

[0201] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0202] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers for proteins and / or nucleic acid provided in the Tables or elsewhere, are incorporated by reference in its entirely.

[0203] The scope of the claims should not be limited by the preferred embodiments and examples but should be given the broadest interpretation consistent with the description as a whole.

Claims

CLAIMS1. An immunoglobulin that binds an IZIIMO protein, the immunoglobulin comprising a heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3 and optionally a signal peptide, wherein the amino acid sequences of saidCDRs are: a)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 2); andWVSTINNDGSIANCDR-H3 (SEQ ID NO: 3);TIGIKNTWRP b)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 4); andWVSAITSGGGSTTCDR-H3 (SEQ ID NO: 5);APGTYHSGPIPD c)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 6); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 7);APGESPAFDGLVEVMD d)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 8); andWVSTINSGGGSTSCDR-H3 (SEQ ID NO: 9); orAPESATSAQAMANIPD e)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 10); andWVSAINSDGGSTSCDR-H3 (SEQ ID NO: 11);APGRDSDYDIIPD f)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 12); andWVSIINSGGASTYCDR-H3 (SEQ ID NO: 13);APEGNPGPIATIPD9)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 14); andWVSTINSGGGKTSCDR-H3 (SEQ ID NO: 15);EPGYQRRYEYD h)CDR-H1 (SEQ ID NO: 16);SSYVMGCDR-H2 (SEQ ID NO: 17); andWVSTITSDGTSARCDR-H3 (SEQ ID NO: 18);APEEKIPIASIPE iCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 19); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 20);APGDSREHGSTWYAI PD jCDR-H1 (SEQ ID NO: 21);SDYAMTCDR-H2 (SEQ ID NO: 22); andWVSSISSGGGSTRCDR-H3 (SEQ ID NO: 23).DIGWTFNNL k)CDR-H1 (SEQ ID NO: 24);SINTMGCDR-H2 (SEQ ID NO: 25); andLVAIINSGDRTNCDR-H3 (SEQ ID NO: 26);NVDSPLYPGRG lCDR-H1 (SEQ ID NO: 27);SINVMGCDR-H2 (SEQ ID NO: 28); andLVAEITSGDTTNCDR-H3 (SEQ ID NO: 29)NIDSPRYGP mCDR-H1 (SEQ ID NO: 59);SISAMTCDR-H2 (SEQ ID NO: 60); andWVARISSGGTTHCDR-H3 (SEQ ID NO: 61);NGIALTEANGSRRYNA nCDR-H1 (SEQ ID NO: 62);SVNGMGCDR-H2 (SEQ ID NO: 63); andLVARITSGSYTDCDR-H3 (SEQ ID NO: 64);WYTDYVDNDGVQ oCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 65); andWVSTINNGGGSTSCDR-H3 (SEQ ID NO: 66); orAPGDYSDYVLQPDFGPCDR-H1 (SEQ ID NO: 82);SVNGMGCDR-H2 (SEQ ID NO: 83); andMVARMTTGGCDR-H3 (SEQ ID NO: 84).YYLDFVGGEGGSLE2. The immunoglobulin of claim 1 , wherein the CDR sequences are SEQ ID NOs: 1-3; SEQ ID NOs: 1 , 4, and 5; SEQ ID NOs: 1 , 6, and 7; SEQ ID NOs: 1 , 8, and 9; SEQ ID NOs: 1 , 10, and 11 ; SEQ ID NOs: 1 , 12, and 13; SEQ ID NOs: 1 , 14, and 15; SEQ ID NOs: 16-18; SEQ ID NOs: 1 , 19, and 20; SEQ ID NOs: 21-23; SEQ ID NOs: 24-26; SEQ ID NOs: 1 and 65-66; and / or SEQ ID NOs: 27-29.

3. The immunoglobulin of claim 1 or 2, wherein the CDR sequences are SEQ ID NOs:1 and 10-11.

4. The immunoglobulin of claim 1 or 2, wherein the CDR sequences are SEQ ID NOs: 1 and 12-13.

5. The immunoglobulin of claim 1 or 2, wherein the CDR sequences are SEQ ID NOs: 16- 18.

6. The immunoglobulin of claim 1 or 2, wherein the CDR sequences are SEQ ID NOs: 21-23.

7. The immunoglobulin of claim 1 or 2 , wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 30; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 30, wherein the CDR sequences are SEQ ID NOs: 1-3; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1-3.

8. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 31 ; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 31 , wherein the CDR sequences are SEQ ID NOs: 1 and 4-5; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 1 and 4-5.

9. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 32; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 32, wherein the CDR sequences are SEQ ID NOs: 1 , and 6-7; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 6-7.

10. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 33; ii) a polypeptide havingan amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 33, wherein the CDR sequences are SEQ ID NOs: 1 and 8-9; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 8-9.

11. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 34; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 34, wherein the CDR sequences are SEQ ID NOs: 1 and 10-11 ; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 10-11.

12. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 35; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 35, wherein the CDR sequences are SEQ ID NOs: 1 and 12-13; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 12-13.

13. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 36; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 36, wherein the CDR sequences are SEQ ID NOs: 1 and 14-15; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 14-15.

14. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 37; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 37, wherein the CDR sequences are SEQ ID NOs: 16-18; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:16-18.

15. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 38; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 38, wherein the CDR sequences are SEQ ID NOs: 1 and 19-20; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:1 and 19-20.

16. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 39; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 39, wherein the CDR sequences are SEQ ID NOs: 21-23; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 21-23.

17. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 40; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 40, wherein the CDR sequences are SEQ ID NOs: 24-26; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:24-26.

18. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 41 ; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 41 , wherein the CDR sequences are SEQ ID NOs: 27-29; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 27-29.

19. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 56; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 56, wherein the CDR sequences are SEQ ID NOs: 59-61 ; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 59-61.

20. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 57; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identity to SEQ ID NO: 57, wherein the CDR sequences are SEQ ID NOs: 62-64; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 62-64.

21. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 58; ii) a polypeptide having an amino acid sequence with at least 80%, at least 90%, or at least 95% sequence identityto SEQ ID NO: 58, wherein the CDR sequences are SEQ ID NOs: 1 , 65 and 66; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 1 , 65, and 66.

22. The immunoglobulin of claim 1 or 2, wherein the heavy chain variable region comprises at least residues Leu29, Ser30, Asn32, Thr33, Tyr37, Leu47, I le50, Asn52, Ser53, Gly54, Asp55, Arg56, Asn73, Asp98, Ser99, ProlOO, Leu101 , Pro103, Gly106, and Arg107 of SEQ ID NO: 40.

23. An immunoglobulin that binds an IZIIMO protein, the immunoglobulin comprising a plurality of heavy chain variable regions each comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3 and optionally a signal peptide, wherein the amino acid sequences of said CDRs are: a)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 2); andWVSTINNDGSIANCDR-H3 (SEQ ID NO: 3);TIGIKNTWRP b)(SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 4); andWVSAITSGGGSTTCDR-H3 (SEQ ID NO: 5);APGTYHSGPIPD c)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 6); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 7);APGESPAFDGLVEVMD d)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 8); andWVSTINSGGGSTSCDR-H3 (SEQ ID NO: 9); orAPESATSAQAMANIPD e)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 10); andWVSAINSDGGSTSCDR-H3 (SEQ ID NO: 11);APGRDSDYDIIPD f)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 12); andWVSIINSGGASTYCDR-H3 (SEQ ID NO: 13);APEGNPGPIATIPD g)CDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 14); andWVSTINSGGGKTSCDR-H3 (SEQ ID NO: 15);EPGYQRRYEYD h)CDR-H1 (SEQ ID NO: 16);SSYVMGCDR-H2 (SEQ ID NO: 17); andWVSTITSDGTSARCDR-H3 (SEQ ID NO: 18);APEEKIPIASIPE iCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 19); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 20);APGDSREHGSTWYAI PD j)CDR-H1 (SEQ ID NO: 21);SDYAMTCDR-H2 (SEQ ID NO: 22); andWVSSISSGGGSTRCDR-H3 (SEQ ID NO: 23).DIGWTFNNL k)CDR-H1 (SEQ ID NO: 24);SINTMGCDR-H2 (SEQ ID NO: 25); andLVAIINSGDRTNCDR-H3 (SEQ ID NO: 26);NVDSPLYPGRG lCDR-H1 (SEQ ID NO: 27);SINVMGCDR-H2 (SEQ ID NO: 28); andLVAEITSGDTTNCDR-H3 (SEQ ID NO: 29)NIDSPRYGP mCDR-H1 (SEQ ID NO: 59);SISAMTCDR-H2 (SEQ ID NO: 60); andWVARISSGGTTHCDR-H3 (SEQ ID NO: 61);NGIALTEANGSRRYNA nCDR-H1 (SEQ ID NO: 62);SVNGMGCDR-H2 (SEQ ID NO: 63); andLVARITSGSYTDCDR-H3 (SEQ ID NO: 64);WYTDYVDNDGVQ oCDR-H1 (SEQ ID NO: 1);SSYAMSCDR-H2 (SEQ ID NO: 65); andWVSTINNGGGSTSCDR-H3 (SEQ ID NO: 66); and / orAPGDYSDYVLQPDFGP)CDR-H1 (SEQ ID NO: 82);SVNGMGCDR-H2 (SEQ ID NO: 83); andMVARMTTGGCDR-H3 (SEQ ID NO: 84).YYLDFVGGEGGSLE24. The immunoglobulin of claim 23, wherein the immunoglobulin is a bi-specific construct.

25. The immunoglobulin of claim 23 , wherein the immunoglobulin is a bi-paratopic construct.

26. The immunoglobulin of any one of claims 1 to 25, wherein the immunoglobulin further comprises a further domain, optionally a Fc domain, optionally a human Fc domain.

27. An immunoglobulin which competes with the immunoglobulin of any one of claims 1 to 26 for binding to an IZIIMO protein.

28. A nucleic acid molecule encoding the immunoglobulin of any one of claims 1 to 27.

29. A vector comprising the nucleic acid molecule of claim 28.

30. A cell comprising the nucleic acid molecule of claim 28, the vector of claim 29, or expressing the immunoglobulin of any one of claims 1 to 27.31 . A composition comprising the immunoglobulin of any one of claims 1 to 27, the nucleic acid molecule of claim 28, the vector of claim 29, or the cell of claim 30, and a diluent or pharmaceutically acceptable carrier, optionally formulated for intravaginal administration, optionally as a film, intrauterine device, ring or gel.

32. A method of inhibiting binding of a JUNO protein with an IZUMO1 protein, the method comprising contacting the IZUMO1 protein with the immunoglobulin of any one of claims 1 to 27.

33. A method of inhibiting fertilization, the method comprising contacting a IZUMO1 protein with the immunoglobulin of any one of claims 1 to 27.

34. The method of claim 32 or 33, wherein the IZUMO1 protein contacted is expressed on a spermatozoa, the spermatozoa optionally in a subject.

35. The method of claim 34, wherein the subject is a human.

36. The method of any one of claims 32 to 35, wherein the contacting comprises administering the immunoglobulin of any one of claims 1 to 27 or the composition of claim 31 , to the subject, optionally intravaginally or systemically.

37. Use of the immunoglobulin of any one of claims 1 to 27 or the composition of claim 31 as a contraceptive.

38. The use of claim 37, wherein the contraceptive is a human contraceptive.

39. A protein complex comprising an IZUMO1 protein interacting with the immunoglobulin of any one of claims 1 to 27.

40. A method of making the immunoglobulin of any one of claims 1 to 27 comprising: a) introducing the nucleic acid of claim 28 or the vector of claim 29, in a suitable cell or b) culturing the cell of claim 30; and isolating the immunoglobulin.41 . A kit or immunological assay comprising the immunoglobulin of any one of claims 1 to 27, the nucleic acid molecule of claim 28, the vector of claim 29, the cell of claim 30, and / or the composition of claim 31 , and optionally a container or vial.

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