Anti-JUNO immunoglobulins and methods of use thereof

Abstract

Described herein are immunoglobulins that bind a JUNO 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 for example SEQ ID NOs: 1-3; 4-6; 7-9; 10-12; 33-35; 36,11, and 37; 4, 38, and 39; and / or 40,11, and 41. Nucleic acids, vectors, cells, compositions, and methods of use thereof are also described.

Description

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

[0001] This application claims the benefit of priority to Canadian Application No. 3,215,566 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- P72850PC00_Sequence_Listing” (53,000 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 JUNO and the use of these immunoglobulins for inhibiting binding of JUNO proteins with IZUMO1 proteins, in particular 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 need 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. Given the small size of nanobodies, they are predicted to be able to penetrate to the perivitelline space to inhibit sperm-egg fusion. Further, 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 selfadministration. The versatility of nanobodies allows flexibility for drug delivery. Due to thenanobody’s high solubility and stability, they can be developed as an injectables for systemic delivery, and / or intravaginal local drug delivery via a dissolvable vaginal film, 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 a JUNO protein, the immunoglobulin comprising 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);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNS b)CDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDY c)CDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3 (SEQ ID NO: 9);AKPRNIIVDLPTTLDEYEYDY d)CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12);AKFKRDGSIRYLPISLDQYEYDYCDR-H1- SSSSTMG (SEQ ID NO: 33);CDR-H2 FVGFIGWSGEPPY (SEQ ID NO: 34); andCDR-H3 AGRTGTGWASNDWT (SEQ ID NO: 35); f)CDR-H1- STYTMS (SEQ ID NO: 36);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKTARGSSWPVYLDDEYD (SEQ ID NO: 37); g)CDR-H1- SSYAMS (SEQ ID NO: 4);CDR-H2 WVSGVNSNGGSTS (SEQ ID NO: 38); andCDR-H3 TKETNGALMRFSGGYSGR (SEQ ID NO: 39); or h)CDR-H1- SSYPMS (SEQ ID NO: 40);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKMKNQQYYSDYANLDDYEYD (SEQ ID NO: 41).

[0013] An aspect of the disclosure includes an immunoglobulin that binds a JUNO 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);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNS b)CDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDY c)CDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3 (SEQ ID NO: 9); and / orAKPRNIIVDLPTTLDEYEYDY d)CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12).AKFKRDGSIRYLPISLDQYEYDY

[0014] Another aspect of the disclosure includes an immunoglobulin that interacts with at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75,77-84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41-42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of a JUNO protein.

[0015] Another aspect of the disclosure includes immunoglobulin that binds a JUNO protein, the immunoglobulin comprising at 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);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNS b)CDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIWPTSLDEYEYDY c)CDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3 (SEQ ID NO: 9);AKPRNIIVDLPTTLDEYEYDY d)CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12);AKFKRDGSIRYLPISLDQYEYDY e)CDR-H1- SSSSTMG (SEQ ID NO: 33);CDR-H2 FVGFIGWSGEPPY (SEQ ID NO: 34); andCDR-H3 AGRTGTGWASNDWT (SEQ ID NO: 35); f)CDR-H1- STYTMS (SEQ ID NO: 36);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKTARGSSWPVYLDDEYD (SEQ ID NO: 37); g)CDR-H1- SSYAMS (SEQ ID NO: 4);CDR-H2 WVSGVNSNGGSTS (SEQ ID NO: 38); andCDR-H3 TKETNGALMRFSGGYSGR (SEQ ID NO: 39); and / orh)CDR-H1- SSYPMS (SEQ ID NO: 40);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKMKNQQYYSDYANLDDYEYD (SEQ ID NO: 41).

[0016] Another aspect of the disclosure includes a nucleic acid molecule encoding any immunoglobulin described herein.

[0017] Another aspect of the disclosure includes a vector comprising any nucleic acid molecule described herein.

[0018] Another aspect of the disclosure includes a cell comprising any nucleic acid molecule, any vector, or expressing any immunoglobulin described herein.

[0019] Another aspect of the disclosure includes a composition comprising any immunoglobulin, nucleic acid molecule, vector, or cell described herein, and a diluent or pharmaceutically acceptable carrier.

[0020] Another aspect of the disclosure includes a method of inhibiting binding of a JUNO protein with an IZUMO1 protein, the method comprising contacting the JUNO protein with any immunoglobulin described herein.

[0021] Another aspect of the disclosure includes a method of inhibiting fertilization, the method comprising contacting a JUNO protein with any immunoglobulin described herein.

[0022] Another aspect of the disclosure includes use of any immunoglobulin or composition described herein as a contraceptive.

[0023] Another aspect of the disclosure includes a protein complex comprising a JUNO protein interacting with one or more of the immunoglobulins described herein.

[0024] Another aspect of the disclosure includes a crystal of any protein complex described herein.

[0025] Accordingly, another aspect of the disclosure includes a screen for identifying an inhibitor that binds a JUNO protein at a contraceptive interface, the method comprising: contacting the JUNO protein with one or more putative inhibitors, determining whether the putative inhibitor interacts with at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acidresidues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41- 42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of the JUNO protein. Interacting with one or more of the amino acid residues identifies the putative inhibitor as a candidate inhibitor or inhibitor of JUNO protein at a contraceptive interface.

[0026] Another aspect of the disclosure includes a kit or immunological 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.BRIEF DESCRIPTION OF DRAWINGS

[0027] An embodiment of the present disclosure will now be described in relation to the drawings in which:

[0028] Figs. 1A-B are graphs showing results of a zona-free human in vitro fertilization functional assay of anti-JUNO nanobodies. Fertilization index is defined as the number of sperm heads in the oocyte. Numbers above the error bars refer to the number of oocyte replicates performed.

[0029] Fig. 2 is a set of 3D surface representation of the human JUNO structure comparing the anti-JUNO nanobody binding footprint on JUNO. The natural interface footprint of IZUMO1 on JUNO is shown for comparison.

[0030] Fig. 3 is a schematic showing interaction of an oocyte and a spermatocyte via JUNO - IZUMO1 proteins. JUNO contains a glycophosphatidylinositol (GPI) anchor to the oocyte membrane, while IZUMO1 is attached to the sperm membrane via a transmembrane helix.

[0031] Fig. 4 is a schematic showing the size differences of a human IgG, a camelid IgG, and a nanobody. Due to the small size of nanobodies, they have the advantage of potentially being able to penetrate through the egg zona pellucida layer.

[0032] Fig. 5 is a flow chart and schematic showing the steps of the general nanobody discovery platform: purified JUNO is used as the antigen to immunize camelids (llama or alpaca), peripheral blood is isolated to isolate the immunoglobulin genes for phage display selection by ELISA, identified phages are cloned and expressed for off-rate ranking and selection of anti-JUNO nanobodies.

[0033] Fig. 6 is a schematic of the biophysical steps for expressing, purifying and biophysically and structurally characterizing the top selected nanobodies.

[0034] Figs. 7A-B are graphs that show the BLI sensorgram and Kd results of various anti-JUNO nanobodies binding to human JUNO. Nanobodies of various concentrations (500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM) were allowed to associate (120 seconds) with immobilized biotin-JUNO immobilized, before the biosensor was returned to the kinetic buffer for dissociation (120 seconds).

[0035] Figs. 8A-B are graphs depicting in Fig. 8A concentration vs % inhibition curves of anti-JUNO nanobodies, measured using BLI and fitted using a logistic dose-response function, and in Fig. 8B, the inhibitory concentration at 50% (IC50 values) of various nanobodies as shown in Fig. 8A. IC50 (measured in nM) was found for each trial at the nanobody that produced 50% inhibition.

[0036] Fig. 9 is a graph depicting the results of melting temperature (Tm) stability analysis of anti-JUNO nanobodies. The Tm is defined as the point at which the protein is 50%:50% folded / unfolded. Higher Tm values indicate a more stable nanobody.

[0037] Figs. 10A-C are co-complexed crystal structures of anti-JUNO nanobodies interacting with JUNO, Fig.lOA shows a ribbon diagram of Nb11 interacting with JUNO, Fig. 10B shows a ribbon diagram of Nb3 interacting with JUNO and Fig 10C shows a ribbon diagram of the natural interaction of JUNO and IZUMO1 as a comparison.

[0038] Fig. 11A-B are representations of the binding interface between anti-JUNO nanobody and JUNO. Specific anti-JUNO nanobody and JUNO amino acids at the interface are shown. In Fig 11A and 11 B, Nb11-JUNO and Nb3-JUNO are shown, respectively.

[0039] Fig. 12 shows a schematic of a ternary complex comprising JUNO and two nanobodies binding to different epitopes (bi-paratopic).

[0040] Figs. 13A-B shows A) a SEC chromatogram of a ternary complex comprising Nb4 and Nb11 and JUNO. The first eluted peak has an estimated size of 52.13 kDa, which corresponds to a Nb4-Nb11-JUNO complex, B) results of SDS PAGE analysis of the ternary complex. Peak l contains Nb4, Nb11 and JUNO, confirming the formation of the Nb4-Nb11-JUNO complex.

[0041] Fig. 14A-I depicts a schematic of the expression and purification workflow and design (Fig. 14A) and electrospray ionization mass spectrometry (Figs. 14B-I). Nanobodies are cloned into a pET-22b vector with a pelB signal peptide and C-terminal 10x-His and thrombin cleavage site for expression in BL21(DE3) cells. Nanobodies are purified by Ni-NTA affinity and size exclusion chromatography, and characterized by SDS-PAGE and mass spectrometry. Massspectra are shown for the various nanobodies. Peaks shown in the spectra illustrate the molecular weight (in Daltons) of the nanobody.

[0042] Figs. 15A-D: Representative BLI sensorgrams showing the effects of physical stress on various nanobodies e.g., Fig. 15A) nanobodies were stressed at 60° C for 24 hours, Fig. 15B) stressed at 30°C for 30 days; Fig. 15C) stressed at low pH (pH 3.7); and Fig. 15D) stressed at 4°C for 30 days. All BLI experiments were completed in triplicates. (LIT04 refers to Nb4-25147, UT11 refers to Nb11-25187, UT03 refers to Nb3-25110, UT09 refers to Nb9-25194).

[0043] Figs. 16A-B: A) Effects of the physical stress on nanobody binding affinities. The values presented in the table represent a fold change in binding of nanobodies to JUNO between physically stressed and untreated nanobodies. B) Kd affinity values as measured by BLI of physically stressed and untreated nanobodies. Circle, square, upward pointing triangle, and downward pointing triangle show the mean of UT03, UT04, UT09, and UT11 , respectively. The error bars show SEM (n=3). (UT04 refers to Nb4-25147, UT11 refers to Nb11-25187, UT03 refers to Nb3-25110, UT09 refers to Nb9-25194).

[0044] Fig. 17: Fluorescence microscopy of zona-free human oocytes inseminated with human sperm in the presence of 10 .g / mL anti-JUNO nanobodies. DNA-specific fluorophore Hoechst 33342 is used to detect for the presence of nuclei. The control reaction with no nanobodies revealed the detection of multiple nuclei from multiple sperm-egg fusion events. The Fl numbers listed in the top right corner of each micrograph reports the number of sperm heads that have penetrated into the oocyte. Fertilization index is defined as the number of sperm heads in the oocyte.

[0045] Fig. 18: Aggregation studies of anti-JUNO nanobodies using the StarGazer II system. Static light scattering measurements as a function of a temperature gradient is measured. The Tagg is defined as the temperature at which there is onset of aggregation. All anti-JUNO nanobodies displayed a Tagg better than 62°C. Experiments are performed in triplicate.

[0046] Fig. 19: Ribbon diagram depicting the interaction of Nb11-25187 and Nb4-25147 to human JUNO.

[0047] Fig. 20: 3D surface representation of the human JUNO structure comparing the anti-JUNO Nb11-25187 and Nb4-25147 nanobody binding footprint on JUNO in the Nb11-Nb4- JUNO ternary co-complex structure.

[0048] Fig. 21 : Immunostaining of JUNO in human oocytes. JNb3, JNb4 and JNb11 penetrate the zona pellucida and accumulate in the perivitelline space. Secondary anti-VHH antibody and an anti-IZUMO1 nanobody are negative controls and show no appreciable staining.

[0049] Fig. 22: Binding affinity of JUNO nanobodies to JUNO after stress tests, p: Sample precipitated after stress test.

[0050] Fig. 23: Aggregation temperature of JUNO nanobodies after stress tests. *: Tagg cannot be determined because there is insufficient sample.

[0051] Fig. 24: Bispecific and biparatopic nanobody strategies to improve affinity. Two nanobodies will be linked to form either bispecific or biparatopic nanobodies that are expected to have increased binding affinity and potency.

[0052] Fig. 25: Binding affinities of bispecific and biparatopic nanobodies. Digital SPR measured binding affinities of single JNb3 and JNb4 nanobodies, bispecific JNb3-(G4S)4-JNb3, JNb3-(G4S)5-JNb3, JNb4-(G4S)4-JNb4, and JNb4-(G4S)5-JNb4, and biparatopic JNb3-(G4S)4- JNb4 and JNb3-(G4S)s-JNb4 nanobodies. Short linker = (648)4 which isGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42) and Long linker = (G4S)5which is GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43). All bispecific and biparatopic nanobodies have subnanomolar or picomolar affinities.

[0053] Fig. 26 is a schematic representation of nanobody JNb4.

[0054] Fig. 27 is a graph showing % solubility and Tagg of mutated JNb4 nanobodies. DM= double mutant of F28T and W99G.

[0055] Fig. 28 is a graph showing Kd of non-mutated and mutated JNb4 nanobodies.DETAILED DESCRIPTIONDefinitions

[0056] 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.

[0057] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those ofordinary 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.).

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 unstatedfeatures, 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.

[0063] 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.

[0064] 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.

[0065] 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."

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The term “nanobody” and variations thereof (e.g., VHH etc), refer to antigenbinding polypeptides 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 which are referred as JNb# or Nb# refer to the same nanobody (e.g., JNb4 and Nb4 and Nb4-25147 all refer to the same nanobody).

[0071] 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 liquidfiller, diluent or encapsulating material. See, for example, U.S. Pat. No. 5,580,852, herein incorporated by reference.

[0072] The term “interacts” as used herein with respect to two or more proteins (e.g., an immunoglobulin and a JUNO protein or a JUNO protein and an IZUMO protein), refers to which residues in a protein directly contact the other (or one other) protein.

[0073] The term “contacts” in the context of a digital association refers to an estimation of the binding free energy of a ligand and protein interaction (e.g., whether a molecule such as an inhibitor is likely to bind another molecule (e.g., JUNO)) by calculating intermolecular and / or other forces including affinity binding, solvent and / or steric forces.

[0074] The term “cell” as used herein refers to a single cell or a plurality of cells.

[0075] 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.

[0076] 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).

[0077] 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 sequences are 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 positionsor 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.

[0078] 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 the art, 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 subjectimmunoglobulin 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.

[0079] 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.

[0080] 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.

[0081] The term “pharmaceutically acceptable” means compatible with the treatment of animals, in particular, humans.

[0082] 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.

[0083] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.

[0084] 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., JUNO protein) are linked directly or indirectly e.g., by a linker. A bispecific construct can for example bind two molecules of JUNO protein.

[0085] 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., JUNO protein) are linked directly or indirectly e.g., by linkers. A multispecific construct can for example bind two or more molecules of JUNO protein or the same JUNO protein, which may e.g., increase efficacy.

[0086] 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., JUNO protein) are linked directly or indirectly e.g., by a linker. A biparatopic construct can for example bind the same molecule or two molecules of Juno protein.

[0087] 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., JUNO protein) are linked directly or indirectly e.g., by linkers. A multi-valent construct can for example bind two or more molecules of JUNO protein or the same JUNO protein.

[0088] 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.

[0089] 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".

[0090] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable aswould 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.

[0091] 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.

[0092] Accordingly, an aspect of the disclosure includes an immunoglobulin that binds a JUNO 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);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNSCDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDYCDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3AKPRNIIVDLPTTLDEYEYDY(SEQ I D N0: 9);CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12);AKFKRDGSIRYLPISLDQYEYDY e)CDR-H1- SSSSTMG (SEQ ID NO: 33);CDR-H2 FVGFIGWSGEPPY (SEQ ID NO: 34); andCDR-H3 AGRTGTGWASNDWT (SEQ ID NO: 35); f)CDR-H1- STYTMS (SEQ ID NO: 36);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKTARGSSWPVYLDDEYD (SEQ ID NO: 37); g)CDR-H1- SSYAMS (SEQ ID NO: 4);CDR-H2 WVSGVNSNGGSTS (SEQ ID NO: 38); andCDR-H3 TKETNGALMRFSGGYSGR (SEQ ID NO: 39); or h)CDR-H1- SSYPMS (SEQ ID NO: 40);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKMKNQQYYSDYANLDDYEYD (SEQ ID NO: 41).

[0093] Another aspect of the disclosure includes an immunoglobulin that binds a JUNO protein, the immunoglobulin comprising at least residues V37, Q39, G42, K43, G44, L45, Y94,A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118of SEQ IDNO: 30; at least residues V37, Q39, G42, K43, G44, L45, Y95, A97, R100, N101 , T109, L110,D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 31 ; at least residues V37,Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and W120 of SEQ ID NO: 32, and / or at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 32.

[0094] An aspect of the disclosure includes an immunoglobulin that binds a JUNO protein, the immunoglobulin comprising a heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2, and CDR-H3, wherein the amino acid sequences of saidCDRs are: a)CDR-H1 (SEQ ID NO: 1);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNS b)CDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDY c)CDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3 (SEQ ID NO: 9); orAKPRNIIVDLPTTLDEYEYDY d)CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12).AKFKRDGSIRYLPISLDQYEYDY

[0095] In some embodiments, the immunoglobulin further comprises a signal peptide. In some embodiments, the signal peptide is pelB signal peptide.

[0096] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, 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.

[0097] In some embodiments, JNb4 (SEQ ID NO: 29) comprises a F28T mutation. In some embodiments, the heavy chain variable region comprises a polypeptide having an amino acid sequence of SEQ ID NO: 55.

[0098] In some embodiments, JNb4 (SEQ ID NO: 29) comprises a W99G mutation. In some embodiments, the heavy chain variable region comprises a polypeptide having an amino acid sequence of SEQ ID NO: 56.

[0099] In some embodiments, JNb4 (SEQ ID NO: 29) comprises a F28T and a W99G mutation. In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 57.

[0100] 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 SEQ ID NO: 30, wherein the CDR sequences are SEQ ID NOs: 4-6; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:4-6.

[0101] 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 SEQ ID NO: 31 , wherein the CDR sequences are as set forth in SEQ ID NOs: 7-9; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:7-9.

[0102] 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 SEQ ID NO: 32, wherein the CDR sequences are SEQ ID NOs: 10-12; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 10-12.

[0103] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 25; 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: 25,wherein the CDR sequences are SEQ ID NOs: 33-35; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:33-35.

[0104] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 27; 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: 27, wherein the CDR sequences are SEQ ID NOs: 4, 38-39; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:4, 38-39.

[0105] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 28; 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: 28, wherein the CDR sequences are SEQ ID NOs: 40, 11 , 41; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 40,11 ,41.

[0106] In some embodiments, the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 26; 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: 26, wherein the CDR sequences are SEQ ID NOs: 36, 11 , 37; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 36, 11 , 37.

[0107] In some embodiments, the immunoglobulin is a nanobody. In some embodiments, the immunoglobulin comprises two or more nanobodies. In some embodiments, the immunoglobulin comprises two nanobodies. In some embodiments, the two or more nanobodies bind with different epitopes. In some embodiments, the two or more nanobodies bind with the same epitope. In some embodiments, the two or more nanobodies are the same nanobody. In some embodiments, the two or more nanobodies are different nanobodies. In some embodiments, the two or more nanobodies are linked via linker. In some embodiments, the linker comprises glycine residues. Nanobodies are approximately 10 times smaller than conventional antibodies (e.g., 12-15 kDa vs. 150 kDa).

[0108] The immunogloublins e.g. VHHs, fusions thereof and the like, described can be comprised in, or affixed to a nanoparticle. This can for example be used to increase the number of immunoglobulins that are combined. In an embodiment, the nanoparticle is a gold nanoparticle and the immunoglobulins or fusions thereof are adsorbed thereon. In other embodiments, the immunoglobulins are physically attached to the nanoparticle, for example using NHS-chemistry. The nanoparticles can comprise for example 5, 10, 15, 20, 24, 25 or more or any number ofimmunoglobulins between 1 and 26. The nanoparticles can be comprised in a composition or kit and can be administered in the methods and uses described herein.

[0109] Another aspect of the disclosure includes an immunoglobulin that binds a JUNO protein, the immunoglobulin comprising at 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);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNSCDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDYCDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3AKPRNIIVDLPTTLDEYEYDY(SEQ I D N0: 9);CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12);AKFKRDGSIRYLPISLDQYEYDY e)CDR-H1- SSSSTMG (SEQ ID NO: 33);CDR-H2 FVGFIGWSGEPPY (SEQ ID NO: 34); andCDR-H3 AGRTGTGWASNDWT (SEQ ID NO: 35);f)CDR-H1- STYTMS (SEQ ID NO: 36);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKTARGSSWPVYLDDEYD (SEQ ID NO: 37); g)CDR-H1- SSYAMS (SEQ ID NO: 4);CDR-H2 WVSGVNSNGGSTS (SEQ ID NO: 38); andCDR-H3 TKETNGALMRFSGGYSGR (SEQ ID NO: 39); and / or h)CDR-H1- SSYPMS (SEQ ID NO: 40);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKMKNQQYYSDYANLDDYEYD (SEQ ID NO: 41).

[0110] 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.

[0111] In some embodiments, the immunoglobulin is a bi-specific construct comprising a first heavy chain variable region and a second heavy chain variable region. Any of the immunoglobulins described herein that binds the same or overlapping epitope on JUNO can be linked or fused as one bi-specific construct.

[0112] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111, E112, Y113, E114, Y115, D116, Y117, and W118of SEQ ID NO: 30 and a second heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30.

[0113] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29 and a second heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29.

[0114] In some embodiments, the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising CDR sequences are SEQ ID NOs:4-6.

[0115] In some embodiments, the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising CDR sequences are SEQ ID NOs:1-3.

[0116] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30 and a second heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30.

[0117] In some embodiments, the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising 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: 4-6; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:4-6.

[0118] In some embodiments, the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, 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.

[0119] In some embodiments, the immunoglobulin is a multi-specific construct.

[0120] In some embodiments, the plurality comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 heavy chain variable regions. In some embodiments, the plurality comprises between 2 to 10 heavy chain variable regions. In some embodiments, the plurality comprises 2 heavy chain variable regions.

[0121] In some embodiments, the immunoglobulin has a molecular weight of up to about 170kDa. In some embodiments, the heavy chain variable region has a molecular weight of about 15kDa.

[0122] In some embodiments, the immunoglobulin is a bi-paratopic construct construct comprising a first heavy chain variable region and a second heavy chain variable region. Any of the immunoglobulins described herein that binds different epitopes on JUNO can be linked or fused as one bi-specific construct.

[0123] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30 and a second heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29.

[0124] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising CDR sequences SEQ ID NOs:1-3 and a second heavy chain variable region comprising CDR sequences SEQ ID NOs: 4-6.

[0125] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising CDR sequences SEQ ID NOs: 1-3 and a second heavy chain variable region comprising CDR sequences SEQ ID NOs: 10-12.

[0126] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, 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; and a second heavy chain variable region comprising iv) a polypeptide having an amino acid sequence of SEQ ID NO: 30; v) 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: 4-6; or vi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:4-6.

[0127] In some embodiments, the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and W120 of SEQ ID NO: 32 and a second heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29.

[0128] In some embodiments, wherein the immunoglobulin comprises a first heavy chain variable region comprising i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, 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; and a second heavy chain variable region comprising iv) a polypeptide having an amino acid sequence of SEQ ID NO: 32; v) 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: 10-12; or vi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 10-12.

[0129] In some embodiments, the immunoglobulin is a multi-valent construct.

[0130] In some embodiments, the plurality comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 heavy chain variable regions. In some embodiments, the plurality comprises between 2 to 10 heavy chain variable regions. In some embodiments, the plurality comprises 2 heavy chain variable regions.

[0131] In some embodiments, the immunoglobulin has a molecular weight of up to about 170kDa. In some embodiments, the heavy chain variable region has a molecular weight of about 15kDa.

[0132] In some embodiments, the immunoglobulin further comprises one or more 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: 44). In some embodiments, the linker comprises multiple repeats of the amino acid sequence GSGSGS (SEQ ID NO: 45). In some embodiments, the linker comprises multiple repeats of amino acid sequenceGGGGS (SEQ ID NO: 46). In some embodiments, the linker comprises an amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 42). 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: 43). 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.

[0133] Another aspect of the disclosure includes an immunoglobulin that competes with the immunoglobulin described herein for binding to a JUNO protein.

[0134] In some embodiments, the immunoglobulin further comprises further domain(s) and / or moieties in addition to heavy chain variable regions. 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.

[0135] In some embodiments, the immunoglobulin has a binding affinity for the JUNO protein measured by Kd and the Kd is a value of lower than 48nM when measured in a biolayer interferometry assay.

[0136] In some embodiments, the Kd is a value of 38.5 nM or lower when measured in a biolayer interferometry assay.

[0137] In some embodiments, the Kd is a value of between 38.5 nM and 4.8nM when measured in a biolayer interferometry assay.

[0138] In some embodiments, the Kd is a value of lower than about 30 nM. In some embodiments, the Kd is a value of lower than about 20 nM. In some embodiments, the Kd is a value of lower than about 10 nM.

[0139] In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein. In some embodiments, % inhibition is as measured by an in vitro fertilization assay or BLI assay. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 76% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 77% inhibition. In some embodiments, theimmunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 78% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 79% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 80% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 85% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 90% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 91% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 92% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 93% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 94% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 95% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 96% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 97% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 98% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 99% inhibition. In some embodiments, the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with 100% inhibition. In some embodiments, the immunoglobulin comprises binds more than one epitope. In some embodiments, the immunoglobulin binds with two epitopes.

[0140] In some embodiments, the immunoglobulin is stable at room temperature.

[0141] An aspect of the disclosure includes an immunoglobulin that interacts with at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75, 77- 80, 82-84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41-42, 44, 58- 59, 63, 65-68, 70, 77, and 161-163 of a JUNO protein. In an embodiment, the immunoglobulin interacts with amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 and / or amino acid residues 31 , 41-42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of a JUNOprotein. The amino acid residue numbers are with reference to the amino acid sequence provided by accession number: NM_001199206, residues 1-250. In an embodiment, the amino acid residue identifies for a particular residue number are those as indicated in accession number NM_001199206, e.g., residue 31 is K31.

[0142] In some embodiments, the immunoglobulin interacts with at least amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 of the JUNO protein. In some embodiments, the immunoglobulin interacts with at least amino acid residues 31 , 41-42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of the JUNO protein. In some embodiments, the immunoglobulin interacts with at least amino acid residues 31 , 41-42, 44-45, 58-59, 62-63, 65- 68, 70, 75, 77-84, 87, 145-147, 161-166 and 171-174 of the JUNO protein.

[0143] In some embodiments, the immunoglobulin interacts with at least one of amino acid residues 45, 62, or 81 and one or more of amino acid residues 67, 75, 146, 164, 165, 166, 171 , 173, 174, 31 , 41 , 42, 59, 63, 67, 68, and 70.

[0144] In some embodiments, the immunoglobulin has at least 95% purity or has been purified to at least 95%.

[0145] In some embodiments, the immunoglobulin is the immunoglobulin described herein.

[0146] In some embodiments, the immunoglobulin further interacts with amino acid residues 65 and 91 of the JUNO protein.

[0147] Another aspect of the disclosure includes a nucleic acid molecule encoding the immunoglobulin described herein.

[0148] Another aspect of the disclosure includes a vector comprising the nucleic acid molecule described herein.

[0149] Another aspect of the disclosure includes a cell comprising the nucleic acid molecule described herein, the vector of claim described herein, or expressing the immunoglobulin described herein.

[0150] Another aspect of the disclosure includes a composition comprising the immunoglobulin described herein, the nucleic acid molecule described herein, the vector described herein, or the cell described herein, and a diluent or pharmaceutically acceptable carrier. Due to the high solubility and stability of the immunoglobulins described herein, they canbe developed as an injectables for systemic delivery, and / or intravaginal local drug delivery via a dissolvable vaginal film, ring or gel.

[0151] In some embodiments, the composition comprises any immunoglobulin described herein and a diluent or pharmaceutically acceptable carrier or diluent.

[0152] In some embodiments, the composition is a lyophilized composition for reconstitution.

[0153] In some embodiments, the composition is a contraceptive composition.

[0154] In some embodiments, the composition is formulated is formulated for intravaginal administration. In some embodiments, the composition is formulated as a film, ring or gel.

[0155] In some embodiments, the composition comprises one or more nanoparticle.

[0156] Another aspect of the disclosure includes a method of inhibiting binding of a JUNO protein with an IZUMO1 protein, the method comprising contacting the JUNO protein with the immunoglobulin described herein. JUNO protein is found for example on oocytes and IZUMO1 is found for example on sperm. Accordingly, contacting JUNO protein, includes for example contacting oocytes that express JUNO protein.

[0157] In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein (e.g. present on oocytes) by the IZUMO1 protein (e.g. present on sperm) by at least about 76% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 77% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 78% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 79% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 80% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 85% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 90% inhibition. In someembodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 91% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 92% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 93% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 94% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 95% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 96% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 97% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 98% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by at least about 99% inhibition. In some embodiments, the method comprises contacting the JUNO protein with an amount of the immunoglobulin sufficient to inhibit binding of the JUNO protein with the IZUMO1 protein by 100% inhibition. In some embodiments, % inhibition is as measured by an in vitro fertilization assay or BLI assay, for example as described herein.

[0158] Another aspect of the disclosure includes a method of inhibiting fertilization, the method comprising contacting a JUNO protein with the immunoglobulin described herein. For example, the method comprises the immunoglobulin described herein being present in a composition and the composition being administered to / in a vaginal canal, thereby allowing contact between JUNO protein and the immunoglobulin described herein.

[0159] In some embodiments, the JUNO protein contacted is expressed on an oocyte, the oocyte optionally in a subject.

[0160] In some embodiments, the subject is a human.

[0161] In some embodiments, the contacting comprises administering the immunoglobulin described herein or the composition described herein, to the subject, optionally intravaginally or systemically.

[0162] In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 76% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 77% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 78% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 79% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 80% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 85% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 90% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 91% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 92% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 93% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 94% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 95% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 96% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 97% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 98% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by at least about 99% inhibition. In some embodiments, the immunoglobulin administered, contacting or in a composition, is in a concentration to inhibit fertilization by 100% inhibition. In some embodiments, % inhibition is as measured by an in vitro fertilization assay.

[0163] Another aspect of the disclosure includes use of the immunoglobulin described herein or the composition described herein as a contraceptive or in the manufacture of a contraceptive.

[0164] 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.

[0165] In some embodiments, the contraceptive is a human contraceptive.

[0166] Another aspect of the disclosure includes a protein complex comprising a JUNO protein, interacting with one or more of the immunoglobulins described herein.

[0167] The JUNO protein in the protein complex, can be a fragment of JUNO protein, for example at least 50 amino acids, or any number between 50 and 249, including 50 and 249. For example, the fragment minimally comprises a stretch of amino acids that includes at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 (e.g., D67, S75, F77, H78, C79, G80, L81 , L82, M83, P84, R87, M145, S146, Y147, K163, N164, R165, C166, Q171, C172, L173, P174) and / or ii) amino acid residues 31 , 41-42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 (e.g., K31 , D41 , K42, Y44, E45, L58, T59, W62, E63, H65, L66, D67, V68, P70, N73, F77, Q161, G162, K163).

[0168] In some embodiments, the JUNO protein minimally comprises amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174. In some embodiments, the JUNO protein minimally comprises residues 31 , 41-42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163. In some embodiments, the JUNO protein minimally comprises residues 31 , 41-42, 44-45, 58-59, 62- 63, 65-68, 70, 75, 77-84, 87, 145-147, 161-166, and 171-174.

[0169] In some embodiments, the JUNO protein minimally comprises at least one of amino acid residues 45, 62, or 81 and one or more of amino acid residues 67, 75, 146, 164, 165, 166, 171 , 173, 174, 31 , 41 , 42, 59, 63, 67, 68, and 70.

[0170] It is demonstrated herein that certain amino acid residues of the nanobodies described herein interact with the JUNO protein. Accordingly, in some embodiments, the immunoglobulin, e.g. nanobody described herein, comprises amino acid residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and / or W118 of nanobody Nb3. In some embodiments, the immunoglobulin, e.g. nanobody described herein, comprises amino acid residues V37, Q39, G42, K43, G44, L45, Y95, A97, R100, N101. T109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and / or W118 of nanobodyNb9. In some embodiments, the nanobody described herein, comprises amino acid residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and / or W120 of the nanobody Nb11. In some embodiments, the nanobody described herein, comprises amino acid residues N32, A33, M34, A35, Y37, Q44, R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and / or W103 of nanobody Nb4. The sequences for said nanobodies are found in Table 11. Immunoglobulins such as heavy chain antibodies can comprise the nanobodies or fragments thereof as described herein. Various numbering conventions for CDRs of exist (e.g., IMGT, Kabat, Chothia, Monte Carlo etc.,) and a person skilled in the art would understand how to identify the equivalent position of the residues described herein in a different immunoglobulin based on different numbering conventions. These immunoglobulins are all contemplated herein.

[0171] In some embodiments, the nanobody described herein, comprises amino acid residues N32, A33, M34, A35, Y37, R45, Q44, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and / or W103 of nanobody Nb4 and amino acid residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and / or W120 of nanobody Nb11.

[0172] In some embodiments, the nanobody described herein, comprises amino acid residues N32, A33, M34, A35, Y37, Q44, R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and / or W103 of nanobody Nb4 and amino acid residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and / or W120 of nanobody Nb11.

[0173] In some embodiments, the JUNO protein interacts with residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of an immunoglobulin described herein. In some embodiments, the JUNO protein interacts with residues V37, Q39, G42, K43, G44, L45, Y95, A97, R100, N101 , T109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and / or W118 of the immunoglobulin described herein. In some embodiments, the JUNO protein interacts with residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and / or W120 of the immunoglobulin described herein. In some embodiments, the JUNO protein interacts with residues N32, A33, M34, A35, Y37, Q44, R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , W103 of the immunoglobulin described herein.

[0174] In some embodiments, the JUNO protein interacts with residues N32, A33, M34, A35, Y37, Q44, R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and / or W103 of the immunoglobulin described herein and residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and / or W120 of the immunoglobulin described herein. In some embodiments, the JUNO protein interacts with residues N32, A33, M34, A35, Y37, Q44, R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and / or W103 of the immunoglobulin described herein and residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and / or W120 of the immunoglobulin described herein simultaneously. Another aspect of the disclosure includes a crystal of the protein complex described herein. In some embodiments, the crystal comprises a JUNO protein or a fragment thereof. In some embodiments, the crystal comprises an immunoglobulin described herein or a fragment thereof. The crystal structure is a bound complex of JUNO to the nanobody. This provides a blueprint as to where an immunoglobulin (or other molecule e.g., small molecules or other binders etc.) effective as a contraceptive could target.

[0175] In some embodiments, the crystal comprises characteristics defined in Table 1A, Table 1 B and / or 1C. In some embodiments, the crystal comprises characteristics defined in Table 1A. In some embodiments, the crystal comprises characteristics defined in Table 1 B. In some embodiments, the crystal comprises characteristics defined in Table 10.

[0176] In some embodiments, the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein to a resolution of 1.9 Angstroms, 1.8 Angstroms, or 2.0 Angstroms.

[0177] In some embodiments, the crystal comprises a space group P1 , P2i 2i 2i, or P2i.

[0178] In some embodiments, the crystal comprises a unit cell of dimensions of a=44.4A, b=56.9A, C=66.0A, a=103.7°, p=91.3°, y=90.9°; a=44.lA, b=73.3A, c=115.5A, a=90.0°, p=90.0°, y=90.0°; or a=65.7A, b=43.6A, c=115.3A, a=90.0°, p=103.0°, y=90.0°.

[0179] The structure determined from diffracting the crystals provides a blueprint that explains where, and how a nanobody binds to the JUNO protein and can be used as a roadmap to developing an inhibitor for inhibiting the interaction between JUNO and IZUMO proteins.

[0180] Accordingly, another aspect of the disclosure includes a screen for identifying an inhibitor that binds a JUNO protein at a contraceptive interface, the method comprising: contacting the JUNO protein with one or more putative inhibitors, determining whether the putative inhibitorinteracts with at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41- 42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of the JUNO protein. Interacting with one or more of the amino acid residues identifies the putative inhibitor as a candidate inhibitor or inhibitor of JUNO protein at a contraceptive interface.

[0181] In some embodiments, the method will comprise determining whether the putative inhibitor interacts with at least one of amino acid residues 45, 62, or 81 and one or more of amino acid residues 67, 75, 146, 164, 165, 166, 171 , 173, 174, 31 , 41 , 42, 59, 63, 67, 68, and 70.

[0182] In some embodiments, screen is a computer implemented method, and wherein the method comprises defining a search radius surface in a molecular model of the JUNO protein, the search radius surface comprising coordinates of the amino acid residues of the JUNO protein (e.g., at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41-42, 44- 45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of the JUNO protein) and the contacting comprises identifying if a digital rendering of the one or more putative inhibitors contacts the search radius surface and / or at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41- 42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of the JUNO protein.

[0183] In some embodiments, the method comprises determining if the putative inhibitor interacts with amino acid residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 of the JUNO protein. In some embodiments, method comprises determining if the putative inhibitor interacts with amino acid residues 31 , 41-42, 44-45, 58-59, 62-63, 65-68, 70, 77, and 161-163 of the JUNO protein. In some embodiments, the method comprises determining if the putative inhibitor interacts with amino acid residues 31 , 41-42, 44-45, 58-59, 62-63, 65-68, 70, 75, 77-84, 87, 145- 147, 161-166 and 171-174 of the JUNO protein.

[0184] In some embodiments, it is determined whether the putative inhibitor interacts with at least the amino acid residues provided in Table 10A. In some embodiments, the screen is a computer implemented method, wherein a digital representation of the JUNO protein is contacted with a digital rendering of the one or putative inhibitors. In some embodiments, the digital representation is of the crystal described herein or a digital representation of the protein complex described herein.

[0185] 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 a JUNO protein to a subject, optionally a llama, isolating the immunoglobulin and / or cells expressing the immunoglobulin specific or selective for the JUNO protein.

[0186] 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.

[0187] 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.

[0188] The following non-limiting examples are illustrative of the present disclosure:EXAMPLESExample 1MethodsAntigen production

[0189] The genes corresponding to full-length human IZUMO1 (GenBank accession number: NM_182575, residues 1-350) and human JUNO (GenBank accession number: NM_001199206, residues 1-250) were codon optimized for expression in Drosophila melanogaster and gene synthesized (Integrated DNA Technologies). The DNA sequences encoding the extracellular regions of IZUMO1 (residues 22-254) and JUNO (residues 20-228) with a BiP signal peptide were subcloned into a modified pMT-puromycin expression vector. All protein constructs contain a thrombin cleavage site and 10X-His affinity tag at the C-terminus. The resulting wild-type IZ UM 0122-254, and JUNO20-228 pMT expression plasmids were stablytransfected in Drosophila S2 cells (Invitrogen) using Effectene transfection reagent (Qiagen), according to manufacturer’s protocol.

[0190] 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 CuSO4. 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.) All IZUMO1 and JUNO proteins were 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 and JUNO were 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

[0191] 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.

[0192] Each animal was injected subcutaneously for 6 times on a weekly basis, each time with an emulsion of adjuvant and purified recombinant JUNO protein (about 100 pg per injection per animal). The adjuvant used was Gerbu P. The general health status (eating habit, bodytemperature, etc.) of the animals was monitored daily throughout the immunization period. The animals remained healthy during the whole procedure.Construction of nine independent VHH libraries

[0193] 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) and His6tag (e.g., HHHHHH).Isolation of nanobodies specific against JUNO

[0194] Each of the 3 immune libraries, derived from the animals immunized with human JUNO, was separately panned on solid-phase coated human JUNO (100 pg / ml in 100 mM NaHCOs pH 8.2) for 3 rounds. 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 antigen-specific 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.

[0195] In summary, the panning and ELISA screening of 3 different immune VHH libraries resulted in the identification of 253 different human JUNO-specific Nanobodies. These 253 unique Nanobodies belong to 48 different B-cell lineages (CDR3 groups). Nanobodies (Nbs) belonging to the same CDR3 group recognize the same epitope, but affinity, potency, stability, expression characteristics can be different.Generation of periplasmic extracts (PE)

[0196] 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 1.2 mM of 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

[0197] In order to rank the nanobody binders, off-rate determination was performed using biolayer interferometry (BLI). The biotinylated human JUNO and IZUMO1 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 l 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-JUNO nanobodies

[0198] In order to determine whether the anti-JUNO nanobody binders inhibited the human JUNO-IZUMO1 interaction, a BLI-based interference assay was performed. Biotinylated human JUNO was coated on Octet SA tips, then the coated tips were allowed to interact with PEs containing anti-JUNO Nbs for 600 seconds, until the saturation was reached. During the next step, tips were soaked in a solution containing human IZUMO1 at 500 nM together with the PEs containing exactly the same Nbs, to avoid leaving the epitope exposed. The binding of IZUMO1 on coated JUNO, 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 IZUMO1 at 500 nM were used as references.Orthogonal fluorescence-based screening assay

[0199] As an orthogonal assay to the BLI-interference assay, a second interference assay based on fluorescence was developed. The batch of 50 semi-purified anti-JUNO nanobodies from PE were diluted to a stock of 100 pg / mL in PBS buffer. Tag-removed recombinant JU NO20-228 was biotinylated in a 1 :1 (biotimprotein) molar ratio (EZ-link sulfo-NHS-LC-biotin; Thermo Scientific, cat#A39257) and tag-removed recombinant IZUMO122-254 was 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- JUNO (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-JUNO 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-IZUMOI 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-JUNO and the buffer added in place of nanobodies and fluorescein-IZUMOI and a negative control with the only the addition of biotin-JUNO bait and fluorescein-IZUMOI , 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

[0200] Epitope binning assay

[0201] In order to determine whether the anti-JUNO 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 possiblewith extracts), the tips were transferred to a mix of PEs containing again the first Nb (at the same concentration) 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.Protein expression and purification

[0202] In order to produce larger yields of the nanobodies for biophysical and structural analysis, the top 12 anti-JUNO nanobodies were subcloned for litre-scale expression in E. coli. The DNA sequences encoding the top 12 anti-JUNO and nanobodies were codon optimized for expression in E. coli, gene synthesized and cloned into the pET22b(+) expression vector (Millipore Sigma) with a vector-encoded N-terminal pelB signal peptide for periplasmic expression (GenScript). The protein constructs also include a thrombin cleavage site (LVPR|GS) and a 10xHIS tag at the C-terminus for tag removal and affinity purification, respectively. The resulting pET22b (+) nanobody expression vectors were then transformed into BL21 (DE3) bacterial cells and plated on LB-ampicillin agar plate. Single colony was then inoculated in LB-amp media to grow the pre-culture overnight at 37 °C. 10 mL of overnight culture was inoculated into 1 L of Terrific Broth (TB; supplemented with a final concentration of 100 pg / mL ampicillin and 0.1 % w / v glucose) and grown at 37°C and 180 rpm until an GD600 nm of 0.6-0.9 was reached. Protein expression was then induced by adding a final concentration of 1 mM IPTG and incubating for 4 hrs at 37°C and 180 rpm. The cells were harvested by centrifugation.

[0203] Nanobodies are extracted from the periplasm via osmotic shock lysis. Here, the bacterial pellet was resuspended in 12 mL of TES and incubated in a refrigerated orbital shaker (180 rpm) at 4°C for 1 hour, prior to the addition of 24 mL TES / 4 and continued incubation for 2 hours. The lysed cells were centrifuged and the periplasm-containing supernatant was decanted. Nanobodies were purified from the periplasmic extract by standard metal affinity chromatography using Ni-charged MagBeads (GenScript, cat#L00295). For tag- removed nanobodies, restrictiongrade thrombin (Millipore Sigma cat#69671-1000UN) was added at a concentration of 1 unit thrombin / mg nanobody and dialyzed overnight at 24°C. 10xHis-tagged and tag-removed nanobodies were further purified by isocratic elution on an ENrich SEC 70 10 x 300 size exclusion column (BioRad, cat#7801070). The fractions containing the nanobodies were pooled andconcentrated using an Amicon centrifugal concentrator (3 kDa MWCO, 4mL). The final nanobody products were stored in either PBS-glycerol (10 mM Phosphate pH 7.4, 2.7 mM KCI, 137 mM NaCI, 5% (v / v) glycerol) or HBS-glycerol (10 mM HEPES pH 7.5, 0.15 M NaCI, 5% (v / v) glycerol). The final concentration of the purified nanobody was confirmed by absorbance at 280 nm and using the Beer-Lambert Law and the theoretical extinction coefficient. The identity and purity of the samples were confirmed using a combination of SDS-PAGE analysis and Electrospray Ionization-Mass Spectroscopy (ESI-MS) (Figs. 14A-I).Binding affinities, kinetics, and IC50 of anti-JUNO nanobodies

[0204] The binding affinities (KD) and kinetics (kon, kOff) of nanobodies to recombinant human JUNO were measured using a single-channel BLItz instrument. Purified JUNO was biotinylated using the EZ-link sulfo-NHS-LC-biotin (Thermo Scientific, cat#A39257) with a 1 :1 biotin to nanobody molar ratio, following the manufacturer’s instructions. Excess biotin was removed via overnight dialysis in PBS buffer. The streptavidin-coated (SA) sensors were first hydrated in BLI rehydration buffer (PBS, 0.5 mg / ml BSA and 0.01% (v / v) Tween-20) for at least 10 minutes. Biotinylated JUNO (bait) was diluted in BLI kinetics buffer (PBS, 0.1 mg / ml BSA and 0.01 % (v / v) Tween-20) to a final concentration of 20 pg / ml and immobilized onto a SA-biosensor for 120 seconds. After a 45-second baseline in BLI kinetics buffer, a series of nanobody concentrations (500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM nanobody in BLI kinetics buffer) were used to associate to JUN02o-22s Over 120 seconds. Finally, SA-biosensors were returned to BLI kinetics buffer for 120 seconds of dissociation. A negative control with only BLI kinetics buffer and no biotinylated JUNO loaded onto the SA-biosensor were performed for each nanobody to detect non-specific binding. The sensorgrams were step-corrected, reference-corrected and fit globally to a 1 :1 binding model. The equilibrium dissociation constant (Kd), association (kon) and dissociation (kOff) rate constants and its associated standard errors were calculated using the using BLItzPro data analysis software (v.1.3). All nanobodies were measured in triplicate and results are presented as mean ± standard error of the mean.

[0205] The degree that anti-JUNO nanobodies are able to interfere with human JUNO and IZUMO1 binding (IC50 values) was determined using a BLI-based blocking assay. The biotinylation procedure and BLI rehydration and kinetics buffer are the same as those used in the binding affinity experiments. First, SA-coated biosensor was loaded with biotinylated JUNO (5 gg / mL) for 150 seconds on an eight-channel Octet Red96 instrument. After a 60-second baseline, the SA-biosensor was immersed into various concentrations of nanobodies (between 5 gM to 0.2 nM) over 600 seconds to to allow for saturated binding to JUNO (Association 1). Subsequently,the biosensor was immersed for 600 seconds into a mixture containing nanobody (same nanobody concentration used in Association 1) plus 500 nM of IZLIMO1 for a second association (Association 2). An IZUMO1-only control without the addition of nanobody was performed and used as a standard for 0% inhibition (100% response). The change in binding in the form of wavelength shift was measured for Association 2 (step-corrected, reference-corrected) and % inhibition was calculated using the equation:

[0206] A concentration vs. % Inhibition curve was constructed for each nanobody and fitted using a 4-parameter logistic dose-response function. The IC50 was then determined from the sigmoidal fitted curve at the [Nb] that produced 50% inhibition.

[0207] All BLI KD and IC50 experiments were performed at room temperature (22 °C) and performed either in triplicates or quadruplicates. OriginPro was used for all sigmodal curve fitting, statistical analysis (Mean, SEM), and figure generation.Human in vitro fertilization assay

[0208] Nanobodies with desirable characteristics (high affinity and nanomolar IC50 values) was tested using a zona-free human in vitro fertilization (IVF) assay (10 oocytes per nanobody). 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-JUNO 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. We havesuccessfully used the in vitro zona-free fertilization assay in several previous studies (Jean et al.2019 Hum Reprod 34, 118-26; Barbaux et al. 2020 Sci Rep 10:5335).Ternary complex purification and validation

[0209] In order to determine whether nanobodies from different epitope bins are able to mutually interact with the antigen, ternary complexes of two nanobodies with the JUNO were formed. The Nb11-JUNO complex was produced by incubating tag-removed human JUNO with excess tag-removed Nb11 (1 :1.2 molar ratio) for 1 h on ice before purification on an ENrich SEC 70 10 x 300 column to purify the binary complex. Fractions containing the Nb11-JUNO complex was then incubated with excess tag-removed Nb1 , Nb5, or Nb7 in a 1 :1.2 molar ratio for 1 h on ice and then separated by SEC again. As a control, free nanobody (Nb1 , Nb5, or Nb7) was also purified by SEC. All SEC peaks were analyzed by SDS-PAGE and / or ESI-MS.

[0210] The molecular weight of the Nb11-Nb4-JUNO ternary complex was assessed by multiangle light scattering coupled to size exclusion chromatography (SEC-MALS). Prior to the experiment, a Superdex 75 Increase 10 / 300 column was equilibrated in HBS buffer at 4 °C and the MALS detector was calibrated with monomeric bovine serum albumin (BSA) at 1.56 mg / ml. Binary Nb4-JUNO (0.2 mg) and ternary Nb11-Nb4-JUNO (0.19 mg) complexes were applied onto the Superdex 75 Increase 10 / 300 column in line with a MiniDawn MALS and T-REX detector (Wyatt). The data were processed, and weight-averaged molecular mass was calculated using the Astra software package (Wyatt).Crystallographic structural studies

[0211] In order to understand the mechanism of how the nanobodies inhibit the JUNO- IZUMO1 interface, X-ray crystallographic studies were performed.

[0212] Crystallization and X-ray data collection: To prepare the nanobody-JUNO (Nb- JUNO) complexes for crystallization, tag-removed JUNO was deglycosylated using PNGase F (New England BioLabs, cat#P0704S) at a ratio of 1 iL for 1 mg of nanobody and at 22 °C for 24 h. Deglycosylated JUNO and tag-removed anti-JUNO nanobody were mixed at a molar ratio of 1 :1.2 and incubated on ice for 1 h prior to size exclusion using an ENrich SEC 70 10 x 300 column equilibrated with HBS buffer. The ternary Nb11-Nb4-JUNO complex was formed by mixing deglycosylated JUNO with excess Nb11 and Nb4 in a molar ratio of 1 : 1.2: 1.2, respectively, prior to SEC. All purified Nb-JUN02o-228 complexes were concentrated to 10 mg / mL prior to crystallization.

[0213] Sitting drop vapour diffusion crystallization trials were performed using an Oryx8 crystallization robot (Douglas Instruments) at 22°C with 0.6 iL drops (0.3 iL complex and 0.3 iL mother liquor) in 96-well low profile I ntelliplates (Art Robbins). Commerical sparse matrix screens, JCSG+ (Qiagen), CRYOS (Qiagen), MCSG-1 (Molecular Dimensions), and MCSG-2 (Molecular Dimensions) suites were used for the initial crystal screening.

[0214] Rod-shaped crystals were obtained for Nb11-JUNO in 0.1 M MgCI2, 0.1 M sodium cacodylate pH 6.5, 50% w / v PEG 200. Nb3-JUNO was crystallized in 0.075 M HEPES Sodium salt pH 7.5, 1.125 M LiSO4, and 25% v / v glycerol, while Nb9-JUNO crystals grew in 1.36 M Ammonium Sulfate, 0.085 M MES pH 6.5, 8.5% v / v Dioxane, 15% v / v glycerol. All crystals were cryoprotected and flash-cooled in liquid nitrogen. Multiple native datasets of the binary and ternary nanobody complexes were remotely collected on beamlines AMX (17ID-1) or FMX (17ID-2) at the National Synchrotron Light Source-ll (NSLS-II) at Brookhaven National Laboratory (Upton, NY).

[0215] Structure determination and refinement: All diffraction data were indexed and integrated with the DIALS program package and scaled and merged using AIMLESS from the CCP4 program suite. Crystallographic data collection and final refinement statistics are presented in Tables 1A and B.

[0216] Table 1A: Data collection and refinement statistics of Nb11-JUNO, Nb3- JUNO, and Nb9-JUNO. Generated using phenix.table_one and AIMLESS.Statistics for the highest-resolution shell are shown in parentheses.*The current refinement values are only in-progress and much still needs to be improved. Only for this dataset, the diffraction data statistics were calculated using AIMLESS and the refinement statistics are calculated by phenix.table one.

[0217] Table 1B: Data collection and refinement statistics of Nb11-Nb4-JUNOStatistics for the highest-resolution shell are shown in parentheses.

[0218] Table 1C: X-ray data collection and refinement statistics.*Statistics for the highest resolution shell is shown in parentheses.

[0219] Nb11-JUNO: The initial structure of Nb11-JUNO was determined by molecular replacement (MR) using phenix.phaser and JUNO (PDB: 5F4E) and llama nanobody (PDB: 5NBD) as search models. One clear solution (Top TFZ = 19.6) placed two copies of the Nb11 and JUNO in the asymmetric unit. The structure was initially built using phenix.autobuild, followed by iterative rounds of manual building in Coot and refinement in phenix. refine. A two-fold non- crystallographic symmetry (NCS) restraint was employed at the start of refinement and then relaxed as the model improved. Torsion-angle simulated annealing refinement, starting at 5000 K, and refinement of atomic displacement parameters, with both individual and Translation / Liberation / Screw (TLS) groups were carried out using phenix.refine while optimizing X-ray / stereochemistry weight.

[0220] Nb3-JUNO: The structure of Nb3-JUNO was determined by MR using phenix.phaser and JUNO (PDB: 5F4E) and llama nanobody structures (PDB: 5NBD) as search models. One clear solution (Top TFZ = 20.1) placed one copy of Nb3 and JUNO in the asymmetric unit. The structure was iteratively refined by alternating manual building in Coot and refinement in phenix.refine, using the same strategies as employed for the structure of Nb11-JUNO, without the NCS restraint.

[0221] Nb9-JUNO: The Nb9-JUNO complex structure was determined by MR using phenix.phaser and JUNO (PDB: 5F4E) and llama nanobody (PDB: 5NBD) as search models. One clear solution (Top TFZ = 19.4) placed two copies of the Nb9 and JUNO in the asymmetric unit. The structure was iteratively refined by alternating manual building in Coot and refinement in phenix. refine, using the same strategies as employed for the structure of Nb11-JUNO and Nb3- JUNO, without the NCS restraint.

[0222] Nb 11-Nb4-JUNO: The initial structure of Nb11-JUNO was determined by molecular replacement (MR) using phenix.phaser. JUNO (PDB: 5F4E), anti-JUNO Nb11-25644 from the Nb11-JUNO structure and an AlphaFold predicted structure of Nb4-25568 as search models. One clear solution (Top TFZ = 33.3) placed one copy of Nb11 , Nb4 and JUNO in the asymmetric unit. The structure was initially built using phenix.autobuild, followed by iterative rounds of manual building in Coot and refinement in phenix.refine. Torsion-angle simulated annealing refinement, starting at 5000 K, and refinement of atomic displacement parameters.

[0223] Validation and structure analysis: The stereochemical quality of the refined models was validated using MolProbity and Coot. No residues were identified in disallowed regions of the Ramachandran plot. The final R-values, B-factors, clash-scores, and RMSD bond lengths and angles of all structural models were consistent with other deposited structures determined at similar resolutions, as determined by polygon. phenix. All structural representations were prepared using PyMOL.Biophysical characterization

[0224] To understand the stability, aggregation potential and quality of the nanobodies, biophysical characterization was performed.

[0225] Circular dichroism spectroscopy: Circular dichroism spectroscopy was performed to understand the stability of the nanobodies. Nanobody samples were 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 were 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 was baseline-corrected, averaged over 8 accumulations and converted to mean residue ellipticity (0MRE).

[0226] 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 was selected; this ranged between 200-203 nm depending on the nanobody. Thermal denaturation assays were conducted by increasing the temperature from 25 to 91 °C, with a full CD wavelengthscan (190 to 260 nm) measured every 2 °C. All data were baseline-subtracted and the change in ellipticity was normalized between 0 (folded) and 1 (unfolded). The resultant curve was fit with a non-linear sigmoidal function using GraphPad Prism 9 (GraphPad Software, San Diego, CA) to determine the melting temperature (Tm). All trials were conducted in triplicates and Tmwere reported as mean ± standard error of the mean.

[0227] Differential static light scattering: DSLS was used to determine the aggregation potential of the nanobodies. Nanobodies were 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 was performed using the Stargazer2 DSLS instrument. The data were processed and analyzed using the Stargazer AIR software to calculate the aggregation temperature (Tagg) for each nanobody. All trials were conducted in triplicates and Taggwere reported as mean ± standard error of the mean.

[0228] Kd of stressed nanobodies: Dissociation constants (KD) of stressed anti-JUNO nanobodies were determined via bio-layer interferometry (BLI) and compared to non-stressed samples and the IZUMO1 protein (Figs. 15A-D and 16A-B). Nb3, Nb9, and Nb11 were able to bind the JUNO protein after the four stress tests (4°C for 30 days; 30°C for 30 days, 60°C for 24 hours, and low pH (~3.7)). Nb4 was able to bind the JUNO protein after exposure to 4°C for 30 days; 60°C for 24 hours, and low pH (~3.7). These stressors were based on conditions associated with PCR (e.g., refrigeration at 4°C for 30 days, room temperature (~30°C) for 30 days), conditions associated with use in LMIC (e.g., ones where there is extreme heat - e.g., 60°C for 24 hours), and conditions associated with use in vivo (e.g., low pH such as vaginal pH of ~4).RESULTS

[0229] Discovery of anti-JUNO nanobodies: Llama immunizations performed at VIB (Brussels) resulted in the identification of 253 anti-JUNO nanobodies, belonging to 48 unique CDR3 groups. Nanobody clones for 253 anti-JUNO nanobodies were expressed in the E. coli periplasm and the crude periplasmic extract (i.e. non-purified) were used for off-rate experiments by BLI. Expression level of nanobodies were analyzed by observing the BLI response units (nanometer wavelength shift). Nanobodies that showed a BLI response of at least 0.2 nm were deemed to have sufficient amounts of nanobodies in the periplasmic extract to saturate the target protein / antigen. Overall, the Nbs BLI signals ranged between 0.2-1.5 nm. k-off rates were then used to rank all Nbs. The top 50 anti-JUNO were arbitrarily selected to produce a full 96-well plate of Nbs (Table 2).

[0230] Table 2: Results of Inhibition Assays for 50 anti-JUNO nanobodies.

[0231] The top 15 nanobodies belonging to 8 different epitope binning groups (Groups A- H) were selected based on their k-off rate and ability to block the JUNO-IZUMO1 interaction in an in vitro BLI assay.

[0232] 12 nanobodies were studied, and 7 anti-JUNO nanobodies were identified that expressed well in the periplasms of E. coli cells and displayed a Kd affinity value (ranging between 4.8-38.5 nM) better than the affinity of the natural JUNO to IZUMO1 interaction (Kd= 48-60 nM) (Table 9). These nanobodies were also effective in blocking the JUNO-IZUMO1 interaction in vitro with mean IC50 values between 23.9-80.2 nM (Table 9). The results show that all nanobodies that had a Kd value equivalent or better than the natural affinity of the IZUMO1-JUNO interaction (Kd=48-60 nM) were effective at blocking the IZUMO1-JUNO interaction. Nanobodies from epitope binning groups A, C, and H all had IC50 values better than 50 nM, whereas epitope group E and G had IC50 values worse than 50 nM. This suggests that epitope location / binding is more important than binding affinity strength once a threshold affinity value has been reached.

[0233] Nanobodies were then studied using a human in vitro fertilization assay, performed at Institute Cochin / INSERM (Paris). Four nanobodies within epitope groups C and H incubated at 10 microgram / ml have -80% or better inhibition of human sperm / egg fusion (Nb4-35147, Nb3- 25110, Nb9-25194, Nb11-25187 - see Table 11 for sequence information), and all three nanobodies in epitope group H have >95% inhibition (Figs. 1A-B). Nanobodies that have better than 80% IVF inhibition were selected for further development (Nb4-35147, Nb11-25187, Nb3- 25110, Nb9-25194). Overall, the best anti-JUNO nanobody (Nb9-251194) inhibited at 100%. Nanobodies in epitope binning groups A, E, and G had poorer inhibition profiles (<80% inhibition), thus suggesting that location of nanobody binding is important to inhibition of fertilization. Nb4- 35147, Nb3-25110, Nb9-25194 and Nb11-25187 nanobodies displayed excellent thermal stability (Tm >67C, Tagg >62C) (Table 9). Structural studies of nanobodies in epitope binning group H (Nb3- 25110 and Nb11-25187) revealed that these nanobodies have -60% overlap to the IZUMO1 binding site, thus explaining its effectiveness in blocking IZUMO1 binding (Fig. 2).

[0234] During fertilization, sperm penetrates through the egg zona pellucida (ZP) glycoprotein layer, binds to the egg membrane, before triggering membrane fusion. Sperm protein IZLIMO1 and its egg receptor JUNO are essential in sperm-egg fusion, but much of the downstream mechanism after JUNO-IZUMO1 binding remains unclear. Small-sized nanobodies (variable domains of camelid IgG) targeting JUNO at the sperm-egg synapse as potential reagents for research and fertilization inhibitors were developed, llama nanobodies specific for human JUNO were generated and screened for their potentials to act as blockers or non-blockers of JUNO-IZUMO1 interactions. 8 of the best blocker anti- JUNO nanobodies were further characterized. 7 / 8 Nbs had KD and IC50 values in the nanomolar range; among them, 3 Nbs displayed >90% inhibition of sperm-egg fusion in human in vitro fertilization assays. The structures of the Nb-JUNO complex revealed that the binding epitope of strong inhibitors overlaps with that of IZUMO1 , therefore likely to inhibit JUNO-IZUMO1 binding via direct competition.

[0235] JUNO and IZUMO1 are thought to be attachment receptors as their binding brings the gametes together (Fig. 3). When their binding was disrupted, fertilization was also inhibited, making JUNO and IZUMO1 valuable targets for contraceptive strategies. Furthermore, the downstream processes after JUNO-IZUMO1 binding remained unclear. Reagents that could effectively penetrate the thick zona pellucida layer would be useful for studying events at the sperm-egg synapse.

[0236] These anti-JUNO nanobodies have the advantage of a small size while retaining high binding affinity and specificity6 7(Fig. 4).

[0237] Anti-JUNO Nbs were generated using llama immunization and selected via Phage Display and ELISA. Initial screening categorized the Nbs as either blockers or non-blockers (Fig. 5). The first batch of Nbs prioritized for investigation was the top 12 blocker candidates that may inhibit JUNO-IZUMO1 interaction.

[0238] Of the 12 top blocker candidates (Nb 1-12), 4 did not express well in small-scale expression, so 8 Nbs were used. The sequences of the 8 blocker nanobody candidates were cloned into the bacterial periplasmic expression vector. After expression and purification, the nanobodies were characterized in their binding affinity, inhibition potentials, thermal stability, and in vitro functional effects. The structures of some nanobody-JUNO complexes were solved using X-ray crystallography (Fig. 6).

[0239] Kd Determination: Biolayer interferometry (BLI) detect protein binding response and calculate the binding affinity (KD) from a concentration series (Fig. 7A). 7 out of 8 nanobodieshave a tighter binding affinity to JUNO than IZUM01 (KD = -40-50 nM) (Fig. 7B). Nb3-25110, Nb9-25194 and Nb11-25187 have the slowest koff kinetic rates (Table 4). These are also the three tightest binding affinity nanobodies characterized, and this tight binding is primarily due to a slow off-rate. Also, Nb3-25110, Nb9-25194 and Nb11-25187 all belong to the same epitope binning group, thus suggesting that structural determinants of binding to this epitope may be the key driver for the long off rate and tight binding Kd (Table 4).

[0240] Table 4: kon, kott, and Kd values for anti-JUNO nanobodies

[0241] IC50 Determination: BLI was also used to measure the IC50 of nanobody in inhibiting IZUMO1 binding to JUNO (Fig. 8A). The 7 Nbs that had KD lower than IZUMO1 also showed IC50 in the lower nanomolar range. The Nb that has a weak binding affinity showed a much higher IC50value (Fig. 8B).

[0242] TmDetermination: Measured using CD, all Nbs had Tm above 60°C (Fig. 9). Static light scattering-based thermal denaturation studies using the StarGazer II system revealed that all anti-JUNO nanobodies had Tagg > 62 °C (Fig. 18 and Table 9) (Note: data was not able to be collected for Nb5-25132 because it was unstable).

[0243] Table 5: Summary of anti-JUNO nanobody biophysical measurements (n = 3).

[0244] Human IVF assays incubated human sperm with depellucidated egg and measured the # of sperm nuclei that penetrated the oocyte. Nb3, 9, and 11 (binning group H) showed > 95% inhibition (Figs. 1A-B and 17). Nb 4 from binning group C also showed -80% inhibition.

[0245] Table 6: Inhibition effects of anti-JUNO Nbs in human IVF assay ([Nb] = 10 pg / mL)

[0246] X-Ray Crystallography: Nb-JUNO complex was co-crystalized. Nb11-JllNO, Nb3- JIINO and Nb9-JUNO produced crystals with good X-ray diffraction data (e.g., Tables 1A-C).

[0247] Binding Structure & Epitope Determination: Both Nb11 and Nb3 contact JUNO via its extended CDR3 loop and a portion of its framework region (Figs. 10A-B and 11A-B). Both bind to the same epitope (Fig. 2). Nb11 overlapped with a portion of IZUMO1 and likely to inhibit through direct competition (Fig. 10C). See Table 10A for residues with which each of Nb3, 9, and 11 interact. See Table 10B for residues of each nanobody which interact with JUNO.

[0248] Table 7: Binding StructureCon LacI Surface Salt bridges (lla.b) ft- at H- bonds # of n- bo ds. <300

[0249] Bi-paratopic Nb construct may improve binding affinity and inhibition (Fig. 12). Nb 4 and Nb 11 were incubated together with JUNO to form the ternary complex. The ternary complex was purified through size-exclusion chromatography (SEC) and analyzed via Multi-angle Light Scattering (SEC-MALS) and SDS-PAGE (Fig. 13A-B and Table 8).

[0250] Nb11-25187 and Nb4-25147 do not bind the same epitope on JUNO and thus can be made into a bi-paratopic nanobody (Fig. 19). A comparison of the combined footprint shows that Nb11-25187 and Nb4-25147 bind to primarily different epitopes on human JUNO. While residues D67, F77, K163 are ones that overlap between the binding of Nb11-25187 and Nb4- 25147 to JUNO, these residues do not form extensive interactions between the nanobody and JUNO thus both nanobodies are still able to bind to JUNO at the same time (Fig. 20).

[0251] Table 8: Molecular weight of ternary and binary Nb-JUNO complexes estimated through SEC-MALS

[0252] Anti-JUNO nanobodies with high affinities and strong inhibiting effects have been identified. Structures of Nb-JUNO complexes showed that the strong inhibitors compete directlywith IZLIM01 for bindings site. Formation of ternary complex confirmed that Nb 11 and Nb 4 binds to different epitopes and may be engineered together as a bi-paratopic blocker construct. 3 anti- JIINO nanobodies were identified that bind with nanomolar affinities and inhibit >90% human in vitro fertilization (Figs. 1A-B and 17).

[0253] Table 9: anti-JUNO nanobody biophysical and functional characterization

[0254] Four nanobodies within epitope groups C and H incubated at 10 mg / ml have -80% or better inhibition of human sperm / egg fusion (Nb4-35147, Nb3-25110, Nb9-25194, Nb11- 25187), and all three nanobodies in epitope group H have >95% inhibition (Table 6). Overall, the best anti-JUNO nanobody (Nb9-251194) inhibited at 100%. Nanobodies in epitope binning groups A, E, and G had poorer inhibition profiles (<80% inhibition), thus suggesting that location of nanobody binding is important to inhibition of fertilization. Nb4-35147, Nb3-25110, Nb9-25194 and Nb11-25187 nanobodies displayed excellent thermal stability (Tm>67°C, Tagg>62°C). Structural studies of nanobodies in epitope binning group H (Nb3-25110 and Nb11-25187) revealed that these nanobodies have -60% overlap to the IZUMO1 binding site, thus explaining its effectiveness in blocking IZUMO1 binding (Fig. 2).

[0255] Downselection of nanobodies for lead optimization and profiling: A set of criteria for selection of nanobodies for the lead optimization and profiling studies described herein is defined below. 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 human or hamster / human IVF assays; >60°C Tmand Tagg.

[0256] A number of anti-JUNO nanobodies that are effective in blocking human in vitro fertilization are identified.

[0257] Table 10A: JUNO epitope residues interacting with Nanobodies

[0258] Table 10B: Nanobody Residues which Interact with JUNO.

[0259] Table 11 : Nanobody Sequences and CDRs (His tag in italics and thrombin cleavage site bolded)Example 2

[0260] A screen for identifying an inhibitor that binds a JUNO protein at a contraceptive interface is carried out by contacting the JUNO protein with one or more putative inhibitors, determining whether the putative inhibitor interacts with JUNO in the same location as nanobodies Nb-4, Nb-3, Nb-9 and / or Nb-4 on the JUNO protein. The determination of whether the putative inhibitor interacts with JUNO at the same location as the aforementioned nanobodies can be determined by assessing whether the the putative inhibitors interacts with JUNO at residues 67, 75, 77-84, 87, 145-147, 163-166, and 171-174 of the JUNO protein. This can be done using computer implemented method where a digital representation of the JUNO protein is contacted with a digital rendering of the one or more putative inhibitors and if the digital rendering of the putative inhibitor is digitally shown to interact with the same location as nanobodies Nb-4, Nb-3, Nb-9 and / or Nb-11 on the JUNO protein (e.g., interacting with the same amino acid residues of JUNO as the aforementioned nanobodies) can be predicted to display the same inhibition properties as the nanobod(ies) which interact with the same location on JUNO (e.g., Nb-4, Nb-3, Nb-9 and / or Nb-11). The digital representations can be crystal structures.Example 3

[0261] Methods for manufacturing immunoglobulins using yeast or bacterial expression systems. Immunoglobulins such as nanobody manifold constructs (bivalent, biparatopic, Fc fusions and nanoparticles) are secreted in a yeast expression system (Pichia pastoris), which is the standard in the production of therapeutic nanobodies. Constructs are codon optimized and gene synthesized into the IP-free yeast expression pPICZ vector (Invitrogen), under an AOX1 promoter. Alpha-mating factor signal peptide sequence is inserted prior to the start of the nanobody gene to target the nanobodies for secretion. The expression vector is electroporated into Pichia and grown in a BMGY medium at 30°C and 220 rpm. Nanobody expression is induced with methanol in a BMMY medium. A time-course induction profile is performed to determine expression levels. Nanobodies are purified from the supernatant using Ni-sepharose Excel resin (Cytiva) followed by FPLC size exclusion chromatography (Superdex 200 Increase). For constructs with a Fc domain, these nanobodies are purified using Protein A followed by FPLC size exclusion chromatography.Example 4Immunostaining of JUNO in human oocytes using nanobodies

[0262] To understand whether the anti-JUNO nanobodies are capable of penetrating the zona pellucida glycoprotein layer and accumulate in the perivitelline space, which is the site wheresperm will fuse with the egg, human oocytes were incubated with anti-JUNO nanobodies JNb3, JNb4, and JNb11 at 10 .g / ml. A second incubation of an Alexa Fluor-488 conjugated goat antialpaca IgG VHH domain secondary antibody was used to detect the presence of nanobodies in the perivitelline space. Results show that the anti-JUNO nanobodies can penetrate through the zona pellucida layer.Stress tests

[0263] Thermal, chemical and physical stress tests on anti-JUNO nanobodies were performed and their effects on binding affinity, aggregation and protein recovery were monitored.

[0264] For thermal stress tests, anti-JUNO nanobodies were incubated at -20C for 30 days, 4°C for 30 days, 30°C for 30 days or 60°C for 24 hours. After stresses, anti-JUNO nanobodies were measured for the binding to recombinant JUNO by BLI, their ability to aggregate (Tagg), and the fraction of anti-JUNO nanobody that was lost due to the stress as compared to unstressed samples.

[0265] For chemical stress tests, anti-JUNO nanobodies were incubated in human serum, at pH 3.7 or pH 9.0. After stresses, anti-IZUMO1 nanobodies were measured for the binding to recombinant JUNO by BLI, their ability to aggregate (Tagg), and the fraction of anti-JUNO nanobody that was lost due to the stress as compared to unstressed samples.

[0266] For physical stress tests, anti-JUNO nanobodies were agitated by physical shaking in an orbital shaker at 200 rpm for 2 hours at 30C. After stresses, anti-IZUMO1 nanobodies were measured for the binding to recombinant JUNO by BLI, their ability to aggregate (Tagg), and the fraction of anti-JUNO nanobody that was lost due to the stress as compared to unstressed samples.

[0267] Table 12A: Summary of Stress Test Results on JUNO nanobodies: Binding affinity (Kd) to JUNO. N D. = Not determined.

[0268] Table 12B: Summary of Stress Test Results on JUNO nanobodies: Aggregation temperature (Tagg) of stressed nanobodies. N.D. = Not determined.

[0269] Table 12C: Summary of Stress Test Results on JUNO nanobodies: Percent protein recovery after stress tests. N.D. = Not determined. * = 0%; + = 0-25%; ++ = 25-50%; +++ = 50-75%;++++ = 75-100%.Example 5Formation of biparatopic nanobodies

[0270] In order to improve the potency of the nanobodies, multiple nanobodies can be combined to improve avidity. Here, two anti-JUNO nanobodies (JNb3 and JNb4) were used using two different linkers (GGGGS)4 (SEQ ID NO: 42) or (GGGGS)s (SEQ ID NO: 43) to form either bispecific or biparatopic nanobodies.

[0271] JNb3-(GGGGS)4-JNb3, JNb3-(GGGGS)5-JNb3, JNb4-(GGGGS)4-JNb4, and JNb4-(GGGGS)s-JNb4 bispecific nanobodies were generated, and JNb3-(GGGGS)4-JNb4 and JNb3-(GGGGS)s-JNb4 biparatopic nanobodies were generated. All bispecific and biparatopic nanobodies were expressed and purified from the periplasm of E. coli then measured for binding by digital SPR (Nicoya Alto system). The combination of nanobodies into bispecific or biparatopic formats significantly improved the binding to JUNO. Bispecific and biparatopic nanobodies have subnanomolar to picomolar binding affinities, which represent a significant improvement of single nanobody binding which as in the nanomolar range.Example 6

[0272] JNb4 is less stable and less soluble after heat stress, especially at 30°C incubated for 30 days or in human serum (Table 13). A cluster of aromatic residues on the surface of JNb4 were noticed and it was hypothesized that they may contribute tothe loss of stability after heat stress (Fig. 26).

[0273] Table 13: Solubility of JNb4 under stress conditions

[0274] F28 and W99 residues on JNb4 were mutated and found that F28T mutation increased solubility after the heat stress (Figs. 27 and 28). JNb4 F28T retains binding to JUNO after the stress tests.

[0275] 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.

[0276] 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.

[0277] 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 a JUNO 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:CDR-H1 (SEQ ID NO: 1);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNSCDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDYCDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3AKPRNIIVDLPTTLDEYEYDY(SEQ I D N0: 9);CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12);AKFKRDGSIRYLPISLDQYEYDYCDR-H1- SSSSTMG (SEQ ID NO: 33);CDR-H2 FVGFIGWSGEPPY (SEQ ID NO: 34); andCDR-H3 AGRTGTGWASNDWT (SEQ ID NO: 35);CDR-H1- STYTMS (SEQ ID NO: 36);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKTARGSSWPVYLDDEYD (SEQ ID NO: 37); g)CDR-H1- SSYAMS (SEQ ID NO: 4);CDR-H2 WVSGVNSNGGSTS (SEQ ID NO: 38); andCDR-H3 TKETNGALMRFSGGYSGR (SEQ ID NO: 39); or h)CDR-H1- SSYPMS (SEQ ID NO: 40);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKMKNQQYYSDYANLDDYEYD (SEQ ID NO: 41).

2. An immunoglobulin that binds a JUNO protein, the immunoglobulin comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, W118 of SEQ ID NO: 30; at least residues V37, Q39, G42, K43, G44, L45, Y95. A97, R100, N101. T109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 31 ; at least residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101, S111 , L112, D113, Q114, Y115, E116, Y117, and W120 of SEQ ID NO: 32, and / or at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and / or W103 of SEQ ID NO: 32.

3. The immunoglobulin of claim 1 or 2, 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);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNS b)CDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDY c)CDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3 (SEQ ID NO: 9); orAKPRNIIVDLPTTLDEYEYDY d)CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12).AKFKRDGSIRYLPISLDQYEYDY4. The immunoglobulin of claim 1 , wherein the heavy chain variable region comprises i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, 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, optionally wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 55.

5. The immunoglobulin of claim 1 , 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: 4-6; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:4-6.

6. The immunoglobulin of claim 1 , 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: 7-9; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:7-9.

7. The immunoglobulin of claim 1 , 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: 10-12; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NQs:10-12.

8. The immunoglobulin of any one of claims 1 to 7, wherein the immunoglobulin comprises one or more nanobody.

9. The immunoglobulin of any one of claims 1 to 8, wherein the immunoglobulin has a binding affinity for the JUNO protein measured by Kd and the Kd is a value of lower than 48nM when measured in a biolayer interferometry assay.

10. The immunoglobulin of claim 7, wherein the Kd is a value of 38.5 nM or lower, optionally lower than about 30 nM, optionally lower than about 20 nM, optionally lower than about 10 nM, when measured in the biolayer interferometry assay.

11. The immunoglobulin of claim 9, wherein the Kd is a value of between 38.5 nM and 4.8nM when measured in the biolayer interferometry assay.

12. An immunoglobulin that interacts with at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75, 77-80, 82-84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41-42, 44, 58-59, 63, 65-68, 70, 77, and 161-163 of a JUNO protein.

13. The immunoglobulin of claim 12, wherein the immunoglobulin is the immunoglobulin of any one of claims 1 to 7.

14. The immunoglobulin of claim 12 or 13, wherein the immunoglobulin further interacts with amino acid residues 65 and 91 of the JUNO protein.

15. An immunoglobulin that binds a JUNO protein, the immunoglobulin comprising at plurality of heavy chain variable regions each comprising complementarity determining regionsCDR-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);SVNAMACDR-H2 (SEQ ID NO: 2); andLVAGITNSGPTTCDR-H3 (SEQ ID NO: 3)AASGPWGNS b)CDR-H1 (SEQ ID NO: 4);SSYAMSCDR-H2 (SEQ ID NO: 5); andWVSAINSGGSTSCDR-H3 (SEQ ID NO: 6);AKVRKGGTIVVPTSLDEYEYDY c)CDR-H1 (SEQ ID NO: 7);SNYAMSCDR-H2 (SEQ ID NO: 8); andWVSAINSGGGSTTCDR-H3 (SEQ ID NO: 9);AKPRNIIVDLPTTLDEYEYDY d)CDR-H1 (SEQ ID NO: 10);SNYAMSCDR-H2 (SEQ ID NO: 11); andWVSAINSGGGSTSCDR-H3 (SEQ ID NO: 12);AKFKRDGSIRYLPISLDQYEYDY e)CDR-H1 SSSSTMG (SEQ ID NO: 33);CDR-H2 FVGFIGWSGEPPY (SEQ ID NO: 34); andCDR-H3 AGRTGTGWASNDWT (SEQ ID NO: 35); f)CDR-H1 STYTMS (SEQ ID NO: 36);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKTARGSSWPVYLDDEYD (SEQ ID NO: 37); g)CDR-H1 SSYAMS (SEQ ID NO: 4);CDR-H2 WVSGVNSNGGSTS (SEQ ID NO: 38); andCDR-H3 TKETNGALMRFSGGYSGR (SEQ ID NO: 39); and / or h)CDR-H1- SSYPMS (SEQ ID NO: 40);CDR-H2 WVSAINSGGGSTS (SEQ ID NO: 11); andCDR-H3 AKMKNQQYYSDYANLDDYEYD (SEQ ID NO: 41).

16. The immunoglobulin of claim 15, wherein the immunoglobulin is a bi-specific construct.

17. The immunoglobulin of claim 15 or 16, wherein the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30 and a second heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30.

18. The immunoglobulin of claim 15 or 16, wherein the immunoglobulin comprises a first heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29 and a second heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29.

19. The immunoglobulin of claim 15 or 16, wherein the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising CDR sequences are SEQ ID NOs:4-6.

20. The immunoglobulin of claim 15 or 16, wherein the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising CDR sequences are SEQ ID NOs:1-3.

21. The immunoglobulin of claim 15 or 16, wherein the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30 and a second heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94, A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30.

22. The immunoglobulin of claim 15 or 16, wherein the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising 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: 4-6; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:4-6.

23. The immunoglobulin of claim 15 or 16, wherein the immunoglobulin comprises a first heavy chain variable region and a second heavy chain variable region each comprising i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, 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.

24. The immunoglobulin of any one of claims 1 to 14, wherein the immunoglobulin is a biparatopic construct.

25. The immunoglobulin of claim 24, wherein the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y94,A96, R99, K100, S109, L110, D111 , E112, Y113, E114, Y115, D116, Y117, and W118 of SEQ ID NO: 30 and a second heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29.

26. The immunoglobulin of claim 24, wherein the immunoglobulin comprises a first heavy chain variable region comprising CDR sequences SEQ ID NOs:1-3 and a second heavy chain variable region comprising CDR sequences SEQ ID NOs: 4-6.

27. The immunoglobulin of claim 24, wherein the immunoglobulin comprises a first heavy chain variable region comprising CDR sequences SEQ ID NOs: 1-3 and a second heavy chain variable region comprising CDR sequences SEQ ID NOs: 10-12.

28. The immunoglobulin of claim 24, wherein the immunoglobulin comprises a first heavy chain variable region comprising i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, 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 I D NOs: 1 -3; and a second heavy chain variable region comprising iv) a polypeptide having an amino acid sequence of SEQ ID NO: 30; v) 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: 4-6; or vi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs:4-6.

29. The immunoglobulin of claim 24, wherein the immunoglobulin comprises a first heavy chain variable region comprising at least residues V37, Q39, G42, K43, G44, L45, Y95, A97, K100, R101 , S111 , L112, D113, Q114, Y115, E116, Y117, and W120 of SEQ ID NO: 32 and a second heavy chain variable region comprising at least residues N32, A33, M34, A35, Y37, Q44 R45, E46, L47, G50, 151 , T52, S54, G55, P56, T57, T58, Y59, A60, A61 , Q64, S96, G97, P98, W99, G100, N101 , and W103 of SEQ ID NO: 29.

30. The immunoglobulin of claim 24, wherein the immunoglobulin comprises a first heavy chain variable region comprising i) a polypeptide having an amino acid sequence of SEQ ID NO: 29; 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: 29, wherein the CDRsequences are SEQ ID NOs: 1-3; or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ I D NOs: 1 -3; and a second heavy chain variable region comprising iv) a polypeptide having an amino acid sequence of SEQ ID NO: 32; v) 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: 10-12; or vi) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are SEQ ID NOs: 10-12.

31. The immunoglobulin of any one of claims 1 to 14, wherein the immunoglobulin is a multivalent construct or a multi-specific construct.

32. The immunoglobulin of any one of claims 1 to 31 , wherein the immunoglobulin further comprises i) one or more linker; ii) a further domain, optionally an Fc domain, optionally a human FC domain.

33. The immunoglobulin of any one of claims 1 to 32, wherein the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 80% inhibition.

34. The immunoglobulin of any one of claims 1 to 32, wherein the immunoglobulin inhibits binding of the JUNO protein and / or oocytes with the IZUMO1 protein with at least about 90% inhibition.

35. An immunoglobulin that competes with the immunoglobulin of any one of claims 1 to 34 for binding to a JUNO protein.

36. A nucleic acid molecule encoding the immunoglobulin of any one of claims 1 to 35.

37. A vector comprising the nucleic acid molecule of claim 36.

38. A cell comprising the nucleic acid molecule of claim 36, the vector of claim 37, or expressing the immunoglobulin of any one of claims 1 to 35.

39. A composition comprising the immunoglobulin of any one of claims 1 to 35, the nucleic acid molecule of claim 36, the vector of claim 37, or the cell of claim 38, and a diluent or pharmaceutically acceptable carrier, optionally formulated for intravaginal administration, optionally as a film, ring, intrauterine device, or gel.

40. A method of inhibiting binding of a JUNO protein with an IZUMO1 protein, the method comprising contacting the JUNO protein with the immunoglobulin of any one of claims 1 to 135.

41. A method of inhibiting fertilization, the method comprising contacting a JUNO protein with the immunoglobulin of any one of claims 1 to 35.

42. The method of claim 40 or 41 , wherein the JUNO protein contacted is expressed on an oocyte, the oocyte optionally in a subject.

43. The method of claim 42, wherein the subject is a human.

44. The method of any one of claims 40 to 43, wherein the contacting comprises administering the immunoglobulin of any one of claims 1 to 35 or the composition of claim 39, to the subject, optionally intravaginally or systemically.

45. Use of the immunoglobulin of any one of claims 1 to 35 or the composition of claim 39 as a contraceptive.

46. The use of claim 45, wherein the contraceptive is a human contraceptive.

47. A protein complex comprising a JUNO protein minimally comprising at least one of amino acid residues 45, 62, or 81 and one or more of i) amino acid residues 67, 75, 77-80, 82- 84, 87, 145-147, 163-166, and 171-174 and / or amino acid residues 31 , 41-42, 44, 58-59, 63, 65-68, 70, 77, and 161-163 interacting with one or more of the immunoglobulins of any one of claims 1 to 35.

48. The protein complex of claim 47, wherein the JUNO protein interacts with a first immunoglobulin comprising CDRs SEQ ID NOs: 1-3 and a second immunoglobulin comprising CDRs at set forth in SEQ ID NOs: 10-12.

49. The protein complex of claim 48, wherein the first immunoglobulin comprises a heavy chain variable region comprising an amino acid sequence SEQ ID NO: 13 and the second immunoglobulin comprises a heavy chain variable region comprising an amino acid sequence SEQ ID NO: 20.

50. A crystal of the protein complex of any one of claims 47 to 49.

51. The crystal of claim 50, wherein the crystal comprises characteristics defined in Table 1A, Table 1 B and / or Table 1C.

52. The crystal of claim 50 or 51 wherein, the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein to a resolution of 1 ,9Angstroms, 1 .8 Angstroms, or 2.0 Angstroms.

53. A screen for identifying an inhibitor that binds a JUNO protein at a contraceptive interface, the method comprising: contacting the JUNO protein with one or more putative inhibitors, determining whether the putative inhibitor interacts with JUNO at at least one of amino acid residues 45, 62 or 81 and one or more of i) amino acid residues 67, 75, 77-80, 82- 84, 87, 145-147, 163-166, and 171-174 and / or ii) amino acid residues 31 , 41-42, 44, 58- 59, 63, 65-68, 70, 77, and 161-163 of the JUNO protein.

54. The screen of claim 53, wherein the screen is a computer implemented method, and wherein the method comprises defining a search radius surface in a molecular model of the JUNO protein, the search radius surface comprising coordinates of the amino acid residues of the JUNO protein and the contacting comprises identifying if a digital rendering of the one or more putative inhibitors contacts the search radius surface.

55. A kit or immunological assay comprising the immunoglobulin of any one of claims 1 to 35, the nucleic acid molecule of claim 36, the vector of claim 37, the cell of claim 38, and / or the composition of claim 39, and optionally a container or vial.

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