WO2022044010A1 - Anti-t-cell immunoglobulin and itim domain (tigit) antibodies for the treatment of fungal infections - Google Patents

Abstract

The present invention provides methods of treating fungal infection, in particular Candida, using monoclonal antibodies that recognize human TIGIT protein.

Description

ANTI-T-CELL IMMUNOGLOBULIN AND ITIM DOMAIN (TIGIT) ANTIBODIES FOR THE TREATMENT OF FUNGAL INFECTIONS

FIELD OF THE INVENTION

The invention is in the field of immunotherapy and relates to monoclonal antibodies for treating fungal infections. In particular, the present invention relates to method of treating Candida using antibodies and fragments thereof which bind to the human T-cell immunoglobulin and ITIM domain (TIGIT) protein.

BACKGROUND OF THE INVENTION

Candida is a genus of fungi and is the most common cause of fungal infections worldwide. Many species are harmless commensals or endosymbionts of hosts including humans; however, when mucosal barriers are disrupted or the immune system is compromised, they can invade and cause disease, known as an opportunistic infection. In recent years, fungal infections have become a serious health problem.

Candidiasis can be present as a cutaneous, mucosal or deep-seated organ infection, which is caused by more than 20 types of Candida species, with C. albicans being the most common. Candida normally lives inside the body (in places such as the mouth, throat, gut, and vagina) and on the skin without causing damage. However, in certain patients who are at risk, Candida can enter the bloodstream or internal organs and cause an infection. A Candida bloodstream infection, also called candidemia, is the most common form of invasive candidiasis. In the United States, candidemia is one of the most common causes of bloodstream infections in hospitalized patients, and it often results in long hospital stays and death.

Antifungal medication can treat invasive candidiasis. Certain patients such as those with cancer or bone marrow or organ transplants might receive antifungal medication to prevent invasive candidiasis. Key challenges to the management of candidemia and invasive candidiasis include prevention, early recognition and rapid initiation of appropriate systemic antifungal therapy.

Formation of biofilms, which diminish the accessibility of antifungal agents, selection of spontaneous mutations that increase expression or decreased susceptibility of the target, altered chromosome abnormalities, overexpression of multidrug efflux pumps and the ability to escape host immune defenses are some of the factors that can contribute to antifungal tolerance and resistance. In addition, anti-fungal drugs sometimes lead to harmful side-effects that prevent their use in some patients. Thus, the development of additional therapy approaches is required.

The fungal agglutinin-like sequence (ALS) gene family encodes cell-surface glycoproteins that are involved in adhesion of fungal cells to host and abiotic surfaces. ALS genes and their encoded proteins are best characterized in C. albicans. A binding cavity located within the N-terminal Als domain is responsible for adhesion to host peptide ligands. Another hallmark of C. albicans Als proteins is an often-extensive central domain of tandemly repeated sequences that are rich in serine, threonine, and sometimes proline (Oh et al. (2019), Front. Microbiol. 10:781).

The human TIGIT protein is expressed on all Natural Killer (NK) cells, as well as on other immune cells such as T regulatory (Treg), CD8+ cells and Tumor infiltrating lymphocytes (Stanietsky et al., PNAS. 2009, 106, 17858-17863). The human TIGIT protein recognizes three very well-defined ligands: poliovirus receptor (PVR, CD155), Nectin-3 (PVRL3/CD113) and Nectin2 (PVRL2/CD112) that are expressed on normal epithelia and over-expressed on various tumor cells. The recognition of these ligands leads to the delivery of an inhibitory signal mediated by two motifs present in the cytoplasmatic tail of TIGIT: the immunoreceptor tail tyrosine (ITT)-like and the immunodominant tyrosine based inhibitory (ITIM) motifs (Liu et al., Cell death and differentiation 2013., 20, 456-464; Stanietsky et al., European journal of immunology, 2013. 43, 2138-2150). TIGIT, through its ITIM domain, inhibit NK cytotoxicity leading to immune evasion mechanism of tumor cells.

TIGIT expression on NK cells also serves as the receptor that binds the Fap2 protein of the anaerobic Gram-negative bacterium Fusobacterium nucleatum (F. nucleatum). The interaction between F. Necleatum and TIGIT leads to reduced NK cytotoxic activity. Fusobacteria are often enriched in patients with intestinal inflammation and cancer. It was suggested that F. nucleatum binding to TIGIT facilitates tumor evasion from NK associated cytotoxicity (Gur et al., Immunity. 2015 February 17; 42(2): 344-355), providing an explanation on how bacteria found within tumors, in particular F. nucleatum, promote tumor proliferation and enhance tumor progression (Jobin, Cancer discovery. 2013; 3:384-387; Sears and Garrett, Cell Host Microbe. 2014; 15:317-328). WO 2004/024068 describes agonists and antagonists to the molecule PRO52254, later identified as TIGIT, for treatment of autoimmune diseases and cancer without disclosing actual antibodies.

WO 2006/124667 discloses modulation of the protein zB7Rl (TIGIT) by monoclonal antibodies that block TIGIT binding to its ligand PVR. No binding affinities are provided.

WO 2009/126688 discloses TIGIT, and its ligand PVR, as targets for modulation of immune responses and suggests agonists and antagonists of these proteins for diagnosis and treatment of immune-related and inflammatory diseases.

WO 2015/009856 discloses combinations of programmed death 1 polypeptide (PD-1) antagonists and anti TIGIT antibodies for treatment of cancer and chronic infection.

WO 2016/028656 discloses anti-TIGIT antibodies, as well as use of these antibodies in the treatment of diseases such as cancer and infectious disease.

WO 2017/037707 discloses isolated monoclonal antibodies which bind to the human TIGIT and suitable for use as anti-cancer agents.

WO 2017/030823 discloses anti-TIGIT antibodies, as well as use of these antibodies in the treatment of diseases such as cancer and infectious disease.

There is an unmet need to provide additional and more effective, specific, safe and/or stable agents that alone or in combination with other agents, are useful in treating fungal infections, in particular Candida.

SUMMARY OF THE INVENTION

The present invention provides methods of treating fungal infections by blocking the interaction between specific fungal Agglutinin-Like Sequences (Als) proteins and the mammalian immune cell inhibitory receptor T-Cell immunoglobulin and ITIM domain (TIGIT) present on lymphocytes. The present invention further provides in some embodiments methods of treating fungal infections using antibodies that specifically recognize mammalian TIGIT and inhibit its suppressive activity on lymphocytes such as Natural Killer (NK) cells and T-cells. These antibodies and fragments thereof, characterized by having unique sets of CDR sequences were found to be highly useful in treating fungal infections, in particular Candida. It is now disclosed that, unexpectedly, antibodies against TIGIT are capable of blocking the interaction between specific ALS proteins present on Candida cells and host TIGIT, and prevent the activation of TIGIT. As a result, the immune cells become uninhibited and act against the Candida infection.

Advantageously, the methods of treating Candida described herein provide the use of anti-TIGIT antibodies that manipulate a mammalian host protein and not the microbial protein, which might be susceptible to selective pressures to evolve resistance to a treatment.

The present invention provides, according to an aspect, an isolated monoclonal antibody for use in treating a fungal infection, the antibody is capable of blocking the interaction between mammalian T-cell immunoglobulin and ITIM domain (TIGIT) and a fungal ALS protein selected from the group consisting of Als6, Als7, and Als9.

According to some embodiments, the isolated monoclonal antibody is capable of blocking the interaction between TIGIT and the fungal ALS protein Als9.

The present invention provides, according to an aspect, an isolated antibody which binds to human T-cell immunoglobulin and ITIM domain (TIGIT), or an antibody fragment thereof comprising at least the antigen binding portion, for treating a fungal infection in a subject or a disease or symptom associated with the fungal infection, wherein the isolated antibody or antibody fragment comprises three heavy-chain (HC) complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 7 and three light-chain (LC) CDRs of a light-chain variable region set forth in SEQ ID NO: 8, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence.

According to some embodiments, the fungal infection is caused by a fungal pathogen selected from the group consisting of Candida, Saccharomyces, Pneumocystis, Aspergillus, and Cryptococcus.

According to some embodiments, the fungal infection is candidiasis.

According to some embodiments, the candidiasis is mucosal candidiasis.

According to some embodiments, the candidiasis is invasive candidiasis. According to some embodiments, the candidiasis is caused by a Candida pathogen species selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida auris, and Candida tropicalis.

According to some embodiments, the antibody is for use in treating or preventing a disease or symptoms thereof associated with infection by a Candida fungal pathogen. According to certain embodiments, the Candida is C. albicans.

According to some embodiments, the subject is a mammalian subject. According to specific embodiments, the subject is human.

According to some embodiments, the isolated antibody is a monoclonal antibody (mAb) or a fragment thereof.

According to some embodiments, the isolated monoclonal antibody or fragment comprises the complementarity determining region (CDR) sequences of a monoclonal antibody denoted VSIG9#1 (or Vsig9.01), namely, the three CDR sequences contained in heavy chain variable region set forth in SEQ ID NO:7 and the three CDR sequences contained in light chain variable region set forth in SEQ ID NO:8. Determination of CDR sequences can be made according to any method known in the art, including but not limited to the methods known as KABAT, Chothia and IMGT. A selected set of CDRs may include sequences identified by more than one method, namely, some CDR sequences may be determined using KABAT and some using IMGT, for example.

According to some specific embodiments, the isolated monoclonal antibody or fragment comprises a set of six CDRs wherein heavy chain CDR1 sequence comprising a sequence selected from the group consisting of: GYTFTSYGIS (SEQ ID NO:1), and TSYGIS (SEQ ID NO: 11), heavy chain CDR2 comprising the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NOG), heavy chain CDR3 comprising the sequence: KGPYYTKNEDY (SEQ ID NOG), light chain CDR1 comprising the sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 comprising the sequence: NANSLED (SEQ ID NOG), and light chain CDR3 comprising the sequence: KQAYDVPRT (SEQ ID NOG), and analogs thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the hypervariable region (HVR) sequence. According to some specific embodiments, the isolated monoclonal antibody or fragment comprises heavy chain CDR1 sequence having the sequence GYTFTSYGIS (SEQ ID NO:1), heavy chain CDR2 having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NOG), heavy chain CDR3 having the sequence: KGPYYTKNEDY (SEQ ID NOG), light chain CDR1 having the sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having the sequence: NANSLED (SEQ ID NOG), and light chain CDR3 having the sequence: KQAYDVPRT (SEQ ID NOG), and analogs thereof comprising no more than 5% amino acid substitution, deletion and/or insertion in the hypervariable region (HVR) sequence.

According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises a heavy chain variable region having the sequence: QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSG NTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKGPYYTKNEDYWG QGTILTVSS (SEQ ID NOG), or an analog or derivative thereof having at least 90% sequence identity with the heavy chain variable region sequence.

According to some embodiments, the isolated monoclonal antibody or fragment thereof comprises a light chain variable region having the sequence: DIQMTQSPASLAASVGETVTITCRASEHIYYSLAWYQQKQGKSPQLLIYNANSLED GVPSRFSGSGSGTQYSMKINSMQPEDTATYFCKQAYDVPRT FGGGTKLEIKRADAAPTVS (SEQ ID NOG), or an analog thereof having at least 90% sequence identity with the light chain variable region sequence.

According to a specific embodiment, the isolated monoclonal antibody or fragment thereof comprises the heavy chain variable region sequence: QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYPRSG NTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKGPYYTKNEDYWG QGTILTVSS (SEQ ID NOG), and the light chain variable region sequence:

DIQMTQSPASLAASVGETVTITCRASEHIYYSLAWYQQKQGKSPQLLIYNANSLED GVPSRFSGSGSGTQYSMKINSMQPEDTATYFCKQAYDVPRTFGGGTKLEIKRADAA PTVS (SEQ ID NOG), or an analog thereof having at least 90% sequence identity with the light and/or heavy chain sequence. Analogs and derivatives of the isolated mAb antibodies, and the antibody fragments described above, are also within the scope of the invention. In particular analogs or isolated mAbs or fragment thereof comprising at least one variable region set forth in a sequence selected from the group consisting of: SEQ ID NOs: 7 and 8, are also within the scope of the present invention.

According to some embodiments, the antibody or antibody fragment analog have at least 95% sequence identity with the hypervariable region of the reference antibody sequence, or at least 90% sequence identity with the heavy or light chain variable regions of the reference antibody.

According to certain embodiments, the analog or derivative of the isolated antibody or fragment thereof has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with a variable region of the reference antibody sequence. Each possibility represents a separate embodiment of the invention.

According to some embodiments, the CDRs have at least 91%, at least 92%, at least 93% or at least 94% sequence identity or similarity with those of VSIG9#1.

According to some embodiments, the antibody or antibody fragment according to the invention comprises a heavy chain variable region set forth in SEQ ID NO:7, or an analog having at least 95% sequence similarity with said sequence.

According to some embodiments, the antibody or antibody fragment comprises a light chain variable region set forth in SEQ ID NO: 8, or an analog having at least 95% sequence similarity with said sequence.

According to some embodiments, the antibody or antibody fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO:7 and the light chain comprises SEQ ID NO:8. Analogs of the antibodies or fragments, having at least 95% sequence similarity with said heavy or light chains are also included.

According to some embodiments, the analog has at least 96, 97, 98 or 99% sequence identity with an antibody light or heavy chain variable regions described above. According to some embodiments, the analog comprises no more than one amino acid substitution, deletion or addition to one or more CDR sequences of the hypervariable region, namely, any one of the CDR sequences set forth in SEQ ID NOs: 1-6. According to some embodiments, the amino acid substitution is a conservative substitution. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the antibody or antibody fragment comprises a hypervariable region (HVR) having light and heavy chain regions defined above, in which 1, 2, 3, 4, or 5 amino acids were substituted, deleted and/or added. Each possibility represents a separate embodiment of the invention. According to specific embodiments, the antibody or antibody fragment comprises a hypervariable region having a set of CDR sequences set forth in SEQ ID NOs.: 1-6, in which no more than one amino acid is substituted, deleted or added to at least one CDR sequence. According to some embodiments, the amino acid substitution is a conservative substitution. Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the antibody or antibody fragment is capable of recognizing TIGIT protein expressed on T-cells.

According to some embodiments, the antibody or antibody fragment is capable of recognizing human TIGIT protein expressed on dendritic or NK cells.

According to some embodiments, the antibody or antibody fragment is capable of recognizing human TIGIT protein expressed on T-regulatory cells (Treg).

According to a specific embodiment, the mAb is selected from the group consisting of: non-human antibody, humanized antibody, human antibody, chimeric antibody, bispecific antibody and an antibody fragment comprising at least the antigen-binding portion of an antibody. According to a specific embodiment, the antibody fragment is selected from the group consisting of: Fab, Fab', F(ab')2, Fd, Fd', Fv, dAb, isolated CDR region, single chain antibody (scab), "diabodies", and "linear antibodies". Each possibility represents a separate embodiment of the present invention.

According to some embodiments, the antibody is a bispecific antibody or bispecific antibody fragment, capable of binding to two different epitopes or antigens, wherein at least one is human TIGIT or an epitope thereof. According to some embodiments, the bispecific mAb comprises two different hyper variable regions (HVR), each comprising a different set of CDR sequences.

According to some embodiments the bispecific mAb or fragment comprises the binding domains of two different anti-TIGIT antibodies. Each HVR of a bispecific mAb or fragment according to these embodiments is cable of binding to a different epitope of the human TIGIT protein.

According to some embodiments, the antibody or antibody fragment comprises a framework sequence selected from the group consisting of: mouse IgG2a, mouse IgG2b, mouse IgG3, human IgGl, human IgG2, human IgG3, and human IgG4. Each possibility represents a separate embodiment of the present invention.

According to other embodiments, the antibody is a humanized antibody or the antibody fragment is a fragment of a humanized antibody.

According to some embodiments, the humanized antibody or antibody fragment comprises a framework sequence selected from the group consisting of: human IgGl, human IgG2, human IgG3, and human IgG4. Each possibility represents a separate embodiment of the present invention.

According to yet other embodiments, an antibody conjugate comprising at least one antibody or antibody fragment that recognizes TIGIT and inhibits binding to its ligand is provided wherein said antibody or antibody fragment comprises heavy chain CDR1 having a sequence selected from: GYTFTSYGIS (SEQ ID NO:1), and TSYGIS (SEQ ID NO: 11), heavy chain CDR2 having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NOG), heavy chain CDR3 having the sequence: KGPYYTKNEDY (SEQ ID NOG), light chain CDR1 having the sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having the sequence: NANSLED (SEQ ID NOG), and light chain CDR3 having the sequence: KQAYDVPRT (SEQ ID NOG).

According to some embodiments, the conjugate comprises an antibody or antibody fragment defined above and a detectable probe or an anti-fungal agent or toxin.

According to some embodiments, the polynucleotide encoding the heavy chain variable region sequence of the antibody used in the compositions and methods of the present invention is set forth in SEQ ID NO: 9. According to some embodiments, the polynucleotide encoding to the light chain variable region sequence of the antibody is set forth in SEQ ID NO: 10.

The present invention provides, according to another aspect, a pharmaceutical composition comprising as an active ingredient, the antibody, antibody fragment or analog thereof as described herein for use in treating a fungal infection, the pharmaceutical composition comprises at least one pharmaceutical acceptable excipient, diluent, salt or carrier.

According to some embodiments, the monoclonal antibody or fragment thereof is capable of binding to an epitope within the human TIGIT protein, the antibody (herein identified as VSIG9#1 or Vsig9.01) comprises a heavy chain variable region of SEQ ID NO:7 and a light chain variable region of SEQ ID NO: 8.

According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or an antibody fragment thereof comprising the six CDR sequences: (i) heavy chain CDR1 sequence selected from: GYTFTSYGIS (SEQ ID NO:1), and TSYGIS (SEQ ID NO: 11), heavy chain CDR2 having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NOG), heavy chain CDR3 having the sequence: KGPYYTKNEDY(SEQ ID NOG), light chain CDR1 having the sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having the sequence: NANSLED (SEQ ID NOG), and light chain CDR3 having the sequence: KQAYDVPRT (SEQ ID NOG).

According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a heavy chain variable region having the sequence: (i)

QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYP RSGNTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKGPYYTKNED YWGQGTILTVSS (SEQ ID NOG).

According to some embodiments, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a light chain variable region having the sequence: DIQMTQSPASLAASVGETVTITCRASEHIYYSLAWYQQKQGKSPQLLIYN ANSLEDGVPSRFSGSGSGTQYSMKINSMQPEDTATYFCKQAYDVPRTFGGGTKLE

IKRADAAPTVS (SEQ ID NO:8).

According to a specific embodiment, the pharmaceutical composition comprises a monoclonal antibody or fragment thereof comprising a heavy chain variable region having the sequence:

QVQLQESGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYP RSGNTYYNEKFKGKATLTADKSSSTAYMELSSLTSEDSAVYFCARKGPYYTKNED YWGQGTILTVSS (SEQ ID NO:7), and a light chain variable region having the sequence:

DIQMTQSPASLAASVGETVTITCRASEHIYYSLA WYQQKQGKSPQLLIYNANSLEDGVPSRFSGSGSGTQYSMKINSMQPEDTATYFCK QA YD VPRT FGGGTKLEIKRADAAPTVS (SEQ ID NO:8).

Also provided are pharmaceutical compositions, comprising at least one antibody, antibody fragment or antibody conjugate according to the invention, for use in restoring NK cytotoxicity by inhibiting binding of TIGIT ligand to fungal cells.

According to some embodiments, a method of restoring NK cytotoxicity is provided by inhibiting binding of TIGIT to at least one ligand expressed on fungal cells, comprising administering to a subject in need thereof a pharmaceutical composition comprising at least one antibody, antibody fragment or antibody conjugate that recognizes human TIGIT as described herein.

According to yet another aspect, the present invention provides a method of treating or preventing a disease or symptoms thereof associated with infection by a fungal pathogen, the method comprising administering to a subject in need thereof an antibody or fragment thereof that recognizes human TIGIT as described herein. According to some embodiments, the fungal pathogen is Candida. According to some specific embodiments, the fungal pathogen is selected from consisting of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida auris, and Candida tropicalis. Each possibility represents a separate embodiment of the invention. According to yet another aspect, the present invention provides a method of treating fungal infection comprising administering to a subject in need thereof, a pharmaceutical composition comprising an effective amount of at least one antibody, antibody fragment or conjugate thereof, that recognizes human TIGIT as described herein.

According to some embodiments, the fungal infection is Candida.

According to certain embodiments, the subject is suffering from a Candida infection. According to additional embodiments, the subject is at risk of suffering from Candida infection. Subjects at risk of suffering from Candida include patients suffering from recurrent Candida infections, patients with suppressed immune systems, diabetic patients, patients under treatment with cortisone-related medications and/or broad-spectrum antibiotics, patients suffering from critical illness, patients recovering from abdominal surgery, patients suffering from a malignant disease, patients undergoing hemodialysis, and pregnant patients.

According to some embodiments, the Candida infection is a C. albicans infection. According to some embodiments, the Candida infection is a vaginal Candida infection. According to some embodiments, Candida infection is a mouth or buccal Candida infection. According to additional embodiments, the Candida infection is a gastrointestinal Candida infection. According to additional embodiments, the Candida infection is an invasive candidiasis infection.

According to some specific embodiments, the monoclonal antibody in the administered pharmaceutical composition comprises: heavy chain CDR1 having the sequence: GYTFTSYGIS (SEQ ID NO:1), heavy chain CDR2 having the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NOG), heavy chain CDR3 having the sequence: KGPYYTKNEDY (SEQ ID NOG), light chain CDR1 having the sequence: RASEHIYYSLA (SEQ ID NO:4), light chain CDR2 having the sequence: NANSLED (SEQ ID NOG), and light chain CDR3 having the sequence: KQAYDVPRT (SEQ ID NOG).

According to some embodiments, the method of treating the fungal infection comprises administering or performing at least one additional anti-fungal therapy.

According to some embodiments, the method further comprises administering an anti- fungal agent selected from the group consisting of Fluconazole, Flucytosine, Nystatin, Amphotericin B, Caspofungin, Voriconazole, natamycin, Posaconazole, lactoferrin, Itraconazole, miconazole, econazole, Micafungin, Ketoconazole, Anidulafungin and Terbinafine. Each possibility represents a separate embodiment of the invention.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1D. TIGIT directly binds Candida cells. Figure 1A - Flow cytometry staining of C. albicans SC5314 yeast cells using TIGIT -Ig (Black empty histogram) or a negative control protein (filled grey histogram). One representative experiment out of 5 is presented. Figure IB - Quantification of the results presented in Figure 1A. Averages of 6 experiments are presented. Figure 1C - EEISA results of plate-bound C. albicans cells stained with TIGIT-Ig or a negative control protein. Averages of 3 experiments are presented. Figure ID - Quantification of flow cytometry staining of various Candida species using TIGIT-Ig or a negative control protein. Average of 2 experiments is presented. Significance was tested using Student’s T-test. ns = not-significant, * = p<0.05, ** = p < 0.01.

Figures 2A-2D. C. albicans binding of TIGIT is functional and leads to inhibition of NK and T cells. Figure 2A - Killing assay of C. albicans yeast cells using the YTS NK cell line either expressing TIGIT (YTS TIGIT) or not expressing it (YTS Eco). The YTS cells were either blocked using anti-TIGIT antibodies (black bars) or not blocked (grey bars). Averages of 2- 4 experiments are presented. Figure 2B - Cytotoxicity assay of C. albicans yeast cells using primary NK cells isolated from different human donors. The NK cells were blocked or not using the anti-TIGIT antibodies. Each line represents an independent human donor. Figure 2C - A Diagram depicting the in-vitro T-cell activation model used in Figure 2D. Figure 2D - T cell activation by C. albicans model. CD4+TIGIT+ T cells were isolated from human donors. The T cells were activated using anti-CD3-antibody-coated P815 cells in the presence or absence of C. albicans cells and the presence (black bars) or absence (grey bars) of a TIGIT blocking antibody. A quantification of 3 independent experiments is presented. Significance was tested using Student’s T-test. ns = not-significant, * = p<0.05, ** = p < 0.01. Figures 3A-3C. TIGIT manipulation by C. albicans is an immune-evasion mechanism in- vivo. Figure 3A - Survival of C. albicans infected C57BL/6 mice depleted for NK cells (red line), T cells (blue line), or mock-depleted (black line). Each line represents 10-12 mice from 2 independent experiments. Figure 3B - A diagram depicting the in-vivo model presented in C and examining the effect of TIGIT blockade on C. albicans. Figure 3C - C. albicans burden in the kidney 48 hours post I.V. infection of C57BE/6 mice. The mice were either treated or not with a TIGIT blocking antibody (as depicted in Figure 3B). Kidneys were harvested, processed, and seeded on Sabouraud dextrose agar plates. Error bars represent the standard error. For the survival assay significance was tested using Mantel-Cox log-rank test. For the fungal burden, significance was tested using Mann-Whitney test, ns = not-significant, * = p<0.05.

Figures 4A-4B. TIGIT manipulation by C. albicans affects human clinical disease. Figure 4A - TIGIT activation was assayed using the murine thymoma cell line BW expressing a chimeric TIGIT-z-chain receptor. The BW cells were co-incubated for 48 hours in the presence of the WT C. albicans lab strain SC5314 (red bar) or clinical C. albicans strains isolated from a cohort of human invasive candidiasis patients (black bars for regular strains or gray bars for super-activating strains). TIGIT activation was measured using EEISA for quantification of IL-2 secreted by the activated BW cells. Figure 4B - A box & whiskers graph showing the difference in time to blood clearance for the regular-activating and super- activating C. albicans isolates presented in Figure 4A. The box represents the interquartile range while the whiskers represent the minimum to maximum range. Significance was tested using Student’s T test, ns = not-significant, * = p<0.05. *** = p<0.005.

Figures 5A-5E. Als proteins of C. albicans are fungal TIGIT ligands. Figure 5A - TIGIT activation was assayed using the murine thymoma cell line BW expressing a chimeric TIGIT- z-chain receptor. The BW cells were co-incubated for 48 hours in the presence of the WT C. albicans strain SC5314 or mutant strains deleted for members of the Als protein family. TIGIT activation was measured using ELISA for IL-2 secreted by the activated BW cells. Shown are averages of 3-4 independent experiments relative to the TIGIT activation abilities of the WT strain. Figure 5B - Cytotoxicity assay of C. albicans strain SC5314 or mutant strains deleted for members of the Als protein family using primary NK cells isolated from different human donors. The NK cells were blocked or not blocked using an anti-TIGIT antibody. Each line represents a different human donor. Figure 5C - Flow cytometry staining of BW and BW TIGIT cells using NT-Als9-2-Ig, NT-Als7-Ig, NT-Als6-Ig or a negative control protein, as indicated. One representative experiment out of 4 is presented. Figure 5D - Quantification of the results presented in Figure 5C. Averages of 4 experiments are presented. Figure 5E - A Microscale Thermophoresis experiment analyzing the interactions between fluorescently labeled TIGIT-Ig and either PVR-Ig, NT-Als9-2-Ig, NT-Als7-Ig, or NT-Als6, as indicated. Shown are a graph based on 3-4 experiments and a summary table. Error bars represent the standard error of the mean. Significance was tested using Student’s T test, ns = not-significant, * = p<0.05, *** = p < 0.001.

Figures 6A-6B. Als9-mediated immune evasion can be targeted using immunotherapy in mice. Figure 6A - C. albicans burden in the kidney 48 hours post I.V. infection of C57BL/6 mice. The mice were infected with either ALS9-, ALS7-, or ALS6- deleted fungal cells, or WT cells, and either treated or not with a TIGIT blocking antibody. Kidneys were harvested, processed, and seeded on Sabouraud dextrose agar plates. The presented results are pooled from 2-5 independent experiments. Figure 6B - Survival of mice infected with 6.5x10⁵ CFU/mouse of the WTC. albicans strain SC5314 or a mutant strain deleted for ALS9, ALS7, or ALS6, and either mock treated or treated with a TIGIT -blocking antibody. Each line represents 7-28 mice from 2-5 independent experiments. Error bars represent the standard error. For the fungal burden, significance was tested using Mann-Whitney test. For the survival assay significance was tested using Mantel-Cox log-rank test, ns = not-significant, * = p<0.05, ** = p<0.01.

Figures 7A-7D. Flow cytometry staining using an anti-TIGIT antibody (Black empty histogram) or an isotype control antibody (filled grey histogram). One representative experiment out of 3 is presented. The cell lines stained were YTS Eco (7 A), YTS TIGIT (7B), BW (Figure 7C) or BW-TIGIT (Figure 7D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides monoclonal antibodies specific to the human protein TIGIT, for use in treating fungal infection.

The present invention provides methods of treating or preventing an infection by one or more fungal pathogens, and/or diseases, disorders, or symptoms thereof, which comprise administering a therapeutically effective amount of an antibody against TIGIT as described herein to a subject in need thereof.

Fungal infections include a variety of pathogenic fungal infections including candidiasis, aspergillosis, coccidioidomycosis, histoplasmosis, penicilliosis and infections by Scedosporium, Saccharomyces or Fusarium.

According to specific embodiments, the method is for treating Candida, the method comprises administering to a subject in need thereof, an anti-TIGIT antibody as described herein.

As used herein, the term "candidiasis" refers to a fungal infection due to any type of Candida (a type of yeast).

Agglutinin-like protein 7 or Als7 is a cell surface adhesion protein which mediates both yeast-to-host tissue adherence and yeast aggregation. Plays an important role in the pathogenesis of C. albicans infections. Als7 in some embodiments has the accession number Q5A312 (UniProt).

Agglutinin-like protein 6 or Als6 (synonym ALS97) is a cell surface adhesion protein which mediates both yeast-to-host tissue adherence and yeast aggregation. Plays an important role in the pathogenesis of C.albicans infections. Als6 in some embodiments has the accession number Q5A2Z7 (UniProt).

Agglutinin-like protein 9 or Als9 (synonym ALSU) is a cell surface adhesion protein which mediates both yeast-to-host tissue adherence and yeast aggregation. Plays an important role in the pathogenesis of C.albicans infections. Allele ALS9-2 contributes to endothelial cell adhesion, whereas ALS9-1 does not. Als9 in some embodiments has the accession number A0A1D8PQ86 (UniProt).

TIGIT (also called T cell immunoreceptor with Ig and ITIM domains) is an immune receptor present on some T cells and Natural Killer Cells (NK). The TIGIT, in some embodiments, refers a TIGIT protein, having an accession number selected from the group consisting of: NP_776160.2; Q495A1.1; AAI01290.1; AAI01291.1; AAI01292.1; ACD74757.1; EAW79602.1; and AIC53385.1; or a fragment of any of said TIGIT proteins. Antibodies, or immunoglobulins, comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to a respective heavy chain by disulfide bonds in a "Y" shaped configuration. Proteolytic digestion of an antibody yields Fv (Fragment variable) and Fc (Fragment crystalline) domains. The antigen binding domains, Fab, include regions where the polypeptide sequence varies. The term F(ab')2 represents two Fab' arms linked together by disulfide bonds. The central axis of the antibody is termed the Fc fragment. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH). Each light chain has a variable domain (VL) at one end and a constant domain (CL) at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain (CHI). The variable domains of each pair of light and heavy chains form the antigen-binding site. The domains on the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, joined by three hyper-variable domains known as complementarity determining regions (CDRs 1-3). These domains contribute specificity and affinity of the antigen-binding site.

CDR determination - CDR identification from a given heavy or light chain variable sequence, is typically made using one of few methods known in the art. For example, such determination is made according to the Kabat (Wu T.T and Kabat E.A., J Exp Med, 1970; 132:211-50) or IMGT (Lefranc M-P, et al., Dev Comp Immunol, 2003, 27:55-77).

When the term “CDR having a sequence”, or a similar term is used, it includes options wherein the CDR comprises the specified sequences and also options wherein the CDR consists of the specified sequence.

The antigen specificity of an antibody is based on the hypervariable regions, namely the unique CDR sequences of both light and heavy chains that together form the antigen-binding site.

The isotype of the heavy chain (gamma, alpha, delta, epsilon or mu) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM, respectively). The light chain is either of two isotypes (kappa, K or lambda, found in all antibody classes. The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multi- specific antibodies (e.g., bi-specific antibodies), and antibody fragments long enough to exhibit the desired biological activity, namely binding to human TIGIT and prevent its interaction with the fungal proteins.

Antibody or antibodies according to the invention include intact antibodies, such as polyclonal antibodies or monoclonal antibodies (mAbs), as well as proteolytic fragments thereof, such as the Fab or F(ab')2 fragments. Single chain antibodies also fall within the scope of the present invention.

Antibody Fragments

Antibody fragments comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., Nature 1989, 341, 544-546) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv) (Bird et al., Science 1988, 242, 423-426; and Huston et al., PNAS (USA) 1988, 85,5879-5883); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 6444-6448); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng., 1995, 8, 1057- 1062; and U.S. Pat. No. 5,641,870).

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv).

Single chain antibodies can be single chain composite polypeptides having antigen binding capabilities and comprising amino acid sequences homologous or analogous to the variable regions of an immunoglobulin light and heavy chain i.e. linked VH-VL or single chain Fv (scFv). Techniques for the production of single-chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single-chain antibodies to TIGIT.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. mAbs may be obtained by methods known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature 1975, 256, 495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature 1991, 352, 624-628 or Marks et al., J. Mol. Biol., 1991, 222:581-597.

The design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy (VH) and variable light (VL) genes and the design and cloning of a synthetic DNA sequence optimized for expression in recombinant bacteria are described in Fields et al.

2013, 8(6): 1125-48.

The mAbs for use of the present invention may be of any immunoglobulin class including IgG, IgM, IgE, and IgA. A hybridoma producing a mAb may be cultivated in-vitro or in-vivo. High titers of mAbs can be obtained by in-vivo production where cells from the individual hybridomas are injected intra-peritoneally into pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. mAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

Besides the conventional method of raising antibodies in vivo, antibodies can be generated in vitro using phage display technology. Such a production of recombinant antibodies is much faster compared to conventional antibody production and they can be generated against an enormous number of antigens. Furthermore, when using the conventional method, many antigens prove to be non-immunogenic or extremely toxic, and therefore cannot be used to generate antibodies in animals. Moreover, affinity maturation (i.e., increasing the affinity and specificity) of recombinant antibodies is very simple and relatively fast. Finally, large numbers of different antibodies against a specific antigen can be generated in one selection procedure. To generate recombinant mAbs one can use various methods all based on display libraries to generate a large pool of antibodies with different antigen recognition sites. Such a library can be made in several ways, for example, generating a synthetic repertoire by cloning synthetic CDR regions in a pool of H chain germline genes and thus generating a large antibody repertoire, from which recombinant antibody fragments with various specificities can be selected. Another example is the use of a lymphocyte pool of humans as starting material for the construction of an antibody library. It is possible to construct naive repertoires of human IgM antibodies and thus create a human library of large diversity. This method has been widely used successfully to select a large number of antibodies against different antigens. Protocols for bacteriophage library construction and selection of recombinant antibodies are provided, for example in Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1.

Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody FR sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

For example, US Patent No. 5,585,089 of Queen et al. discloses a humanized immunoglobulin and methods of preparing same, wherein the humanized immunoglobulin comprises CDRs from a donor immunoglobulin and VH and VL region FRs from human acceptor immunoglobulin H and L chains, wherein said humanized immunoglobulin comprises amino acids from the donor immunoglobulin FR outside the Kabat and Chothia CDRs, and wherein the donor amino acids replace corresponding amino acids in the acceptor immunoglobulin H or L chain frameworks.

Also, transgenic mice, or other organisms such as other mammals, can be used to express humanized antibodies.

US Patent No. 5,225,539, of Winter, also discloses an altered antibody or antigen- binding fragment thereof and methods of preparing same, wherein a V domain of the antibody or antigen-binding fragment has the FRs of a first immunoglobulin H or L chain V domain and the CDRs of a second immunoglobulin VH or VL domain, wherein said second immunoglobulin VH or VL domain is different from said first immunoglobulin VH or VL domain in antigen binding specificity, antigen binding affinity, stability, species, class or subclass.

The above-described antibodies can be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by, for example, affinity chromatography .

The invention provides uses of a monoclonal antibody or an antibody fragment comprising an antigen binding domain (ABD) which comprises three CDRs of a light chain and three CDRs of a heavy chain, wherein said ABD has at least 90% sequence identity or similarity with an ABD of a monoclonal mouse antibody comprising a heavy variable chain comprising the amino acid sequence SEQ ID NO:7 and a light variable chain comprising the amino acid sequence SEQ ID NO:8 (herein identified as VSIG9#1). Such antibody may have an ABD domain having at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, at least 99% sequence identity or similarity or 100% sequence identity with corresponding ABD of VSIG9#1. Sequence identity is the amount of amino acids or nucleotides which match exactly between two different sequences. Sequence similarity permits conservative substitution of amino acids to be determined as identical amino acids.

The invention also provides conservative amino acid variants of the antibody molecules according to the invention. Variants according to the invention also may be made that conserve the overall molecular structure of the encoded proteins. Given the properties of the individual amino acids comprising the disclosed protein products, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e. "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. The term “antibody analog” as used herein refers to an antibody derived from another antibody by one or more conservative amino acid substitutions.

The term “antibody variant” as used herein refers to any molecule comprising the antibody of the present invention. For example, fusion proteins in which the antibody or an antigen-binding-fragment thereof is linked to another chemical entity is considered an antibody variant.

Analogs and variants of the antibody sequences are also within the scope of the present application. These include but are not limited to conservative and non-conservative substitution, insertion and deletion of amino acids within the sequence. Such modification and the resultant antibody analog or variant are within the scope of the present invention as long as they confer, or even improve the binding of the antibody to the human TIGIT.

Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain, e.g., aliphatic, aromatic, positively charged, negatively charged. These substitutions may enhance oral bioavailability, penetration into the islets, targeting to specific beta cell populations, immunogenicity, and the like. One of skill will recognize that individual substitutions, deletions or additions to a peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term "human antibody" as used herein refers to an antibody which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art.

The terms "molecule having the antigen-binding portion of an antibody" and “antigen- binding-fragments” as used herein is intended to include not only intact immunoglobulin molecules of any isotype and generated by any animal cell line or microorganism, but also the antigen-binding reactive fraction thereof, including, but not limited to, the Fab fragment, the Fab' fragment, the F(ab')2 fragment, the variable portion of the heavy and/or light chains thereof, Fab mini-antibodies (see e.g., WO 93/15210, US patent application 08/256,790, WO 96/13583, US patent application 08/817,788, WO 96/37621, US patent application 08/999,554), dimeric bispecific mini-antibodies (see Muller et al., FEBS Lett. 1998 Jul 31;432(1-2):45-9) and single-chain antibodies incorporating such reactive fraction, as well as any other type of molecule in which such antibody reactive fraction has been physically inserted. Such molecules may be provided by any known technique, including, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

A humanized antibody, typically has a human FR grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 527:522-525 (1986); Riechmann et al., Nature, 552:323-327 (1988); Verhoeyen et al., Science, 259:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human VH and VL domains to be used in making the humanized antibodies is very important for reducing immunogenicity. According to the so-called "best-fit" method, the sequence of the V domain of a rodent antibody is screened against the entire library of known human -domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 757:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular FR derived from the consensus sequence of all human antibodies of a particular subgroup of H or L chains. The same FR may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three- dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

Pharmacology

In pharmaceutical and medicament formulations, the active agent is preferably utilized together with one or more pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.

Typically, the antibodies and fragments thereof of the present invention comprising the antigen binding portion of an antibody will be suspended in a sterile saline solution for therapeutic uses. The pharmaceutical compositions may alternatively be formulated to control release of active ingredient (molecule comprising the antigen binding portion of an antibody) or to prolong its presence in a patient's system. Numerous suitable drug delivery systems are known and include, e.g., implantable drug release systems, hydrogels, hydroxymethylcellulose, microcapsules, liposomes, microemulsions, microspheres, and the like. Controlled release preparations can be prepared through the use of polymers to complex or adsorb the molecule according to the present invention. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebaric acid. The rate of release of the molecule according to the present invention, i.e., of an antibody or antibody fragment, from such a matrix depends upon the molecular weight of the molecule, the amount of the molecule within the matrix, and the size of dispersed particles.

The pharmaceutical composition of this invention may be administered by any suitable means, such as orally, topically, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, intraarticulary, or parenterally. Ordinarily, intravenous (i.v.) administration is used for delivering antibodies.

It will be apparent to those of ordinary skill in the art that the therapeutically effective amount of the molecule according to the present invention will depend, inter alia upon the administration schedule, the unit dose of molecule administered, whether the molecule is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the molecule administered and the judgment of the treating physician. As used herein, a “therapeutically effective amount” refers to the amount of a molecule required to alleviate one or more symptoms associated with a disorder being treated over a period of time.

The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of a fungal infection, the therapeutically effective amount of the drug may reduce the number of fungi cells; and reduce to some extent one or more of the symptoms associated with the disorder.

The molecules of the present invention as active ingredients are dissolved, dispersed or admixed in an excipient that is pharmaceutically acceptable and compatible with the active ingredient as is well known. Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, or the like and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents.

Typically, the carrier or excipient for a pharmaceutical composition as described herein is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions ensuring sterility, pH, isotonicity, and stability is affected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like.

Pharmaceutical compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampules), or in vials containing several doses and in which a suitable preservative may be added. The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. In some cases, an active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the pharmaceutical compositions may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.

The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease as well as those in which the disease is to be prevented.

The treatment described herein encompasses enhancing the immune response against the fungal infection.

The term "enhancing immune response" refers to increasing the responsiveness of the immune system and prolonging its memory. The pharmaceutical composition according to the present invention may be used to stimulate immune system upon vaccination.

The term "treating” refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms associated with fungal infection amelioration, and other beneficial results.

The term “effective amount” as used herein refers to a sufficient amount of the monoclonal antibody or the antibody fragment that, when administered to a subject will have the intended therapeutic effect. The effective amount required to achieve the therapeutic end result may depend on a number of factors including, for example, the severity of the patient's condition, and whether an additional therapy is administered. The effective amount (dose) of the active agents, in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the subject over time.

Toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the maximal tolerated dose for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending inter alia upon the dosage form employed, the dosing regimen chosen, the composition of the agents used for the treatment and the route of administration utilized among other relevant factors. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. Depending on the severity and responsiveness of the condition to be treated, dosing can also be a single administration of a slow-release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, and all other relevant factors.

The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered enteral] y or parenterally. Enterally refers to administration via the gastrointestinal tract including per os, or sublingually. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. According to some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self- administer a drug, or to have the drug administered by another and/or who provides a patient with a prescription for a drug is administering the drug to the patient.

Antibodies are generally administered in the range of about 0.1 to about 20 mg/kg of patient weight, commonly about 0.5 to about 10 mg/kg, and often about 1 to about 5 mg/kg. In this regard, it is preferred to use antibodies having a circulating half-life of at least 12 hours, preferably at least 4 days, more preferably up to 21 days. Chimeric and humanized antibodies are expected to have circulatory half-lives of up to four and up to 14-21 days, respectively. In some cases, it may be advantageous to administer a large loading dose followed by periodic (e.g., weekly) maintenance doses over the treatment period. Antibodies can also be delivered by slow-release delivery systems, pumps, and other known delivery systems for continuous infusion. According to an additional aspect, the invention provides a method of providing immunity (or immune protection) to a subject in need who has an infection or who is at risk of having an infection by one or more, or two or more, different fungal types, including Candida, Pneumocystis, Aspergillus and Cryptococcus fungal organisms, by administering to the subject an antibody or a fragment thereof as described herein.

The antibodies disclosed herein may be administered in combination with one or more of any other treatment or therapy, e.g., anti-fungal therapies.

The antibodies may be administered in combination with other antibodies or antibody cocktails with anti-fungal activity. According to some embodiments, the antibodies can be administered alone or in combination with a co-agent useful in the prevention and/or treatment of Candida infections.

The antibodies or compositions described herein may be administered alone or in combination with one or more drugs, for examples, one or more drugs having anti-fungal activity (e.g., trimethoprim-sulfamethoxazole, azithromycin-sulfamethoxazole, clarithromycin-sulfamethoxazole, atovaquone, sulfadoxine -pyrimethamine, erythromycin- sulfisoxazole, and dapsone-trimethoprim, as well as intravenous pentamidine and clindamycin-primaquine) .

According to some embodiments the method further comprises administering an anti- fungal agent selected from the group consisting of Fluconazole, Flucytosine, Nystatin, Amphotericin B, Caspofungin, Voriconazole, natamycin, Posaconazole, lactoferrin, Itraconazole, Micafungin, Ketoconazole, Anidulafungin and Terbinafine. Each possibility represents a separate embodiment of the invention.

The term "about" means that an acceptable error range, e.g., up to 5% or 10%, for the particular value should be assumed.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES

Experimental Procedures

Primary Cells and Cell Lines

Primary NK and T cells were isolated from blood donations collected from healthy individuals and under the approval of the institutional Helsinki committee (Helsinki number 0030-12-HMO). Peripheral blood mononuclear cells (PBMCs) were produced from heparin- treated blood after centrifugation in the presence of Lymphoprep (STEMCELL Technologies). NK were then isolated using EasySep human NK cell enrichment kit (STEMCELL Technologies) and co-cultured in U-bottomed 96-well plates with irradiated (6000 RAD) PBMCs from two independent donors (5x10⁴/well per donor) and irradiated (6000 RAD) RPMI-8866 cells (5x10³/well). The cells were grown in DMEM:F12 medium mix (70:30) with 10% human serum (Sigma-Aldrich), 1 mM sodium pyruvate (Biological Industries), 2 mM glutamine (Biological Industries), nonessential amino acids (Biological Industries), 100 U/ml penicillin (Biological Industries), 0.1 mg/ml streptomycin (Biological Industries), 500 U/ml rhIL-2 (PeproTech) and 20 mg/ml PHA (Sigma-Aldrich). The cells were grown in 37°C and 5% CO₂. NK cell identity was validated using dual staining with anti-human-CD56-PE and anti-human-CD3-FITC antibodies (both from BioLegend) and flow cytometry.

T cells were purified similarly up to the enrichment stage. In its place the PBMCs were cultured in similar conditions to the enriched NK cells but in low cell densities in order to get clonal cultures in the different wells. Following recovery and proliferation of the cells each well was stained using anti-human CD4-PE and anti-human TIGIT-APC antibodies (both from BioLegend) and analyzed using flow cytometry. CD4+TIGIT+ clones were further maintained and used for experiments.

Cell lines used in this study were HEK293T cells, YTS Eco cells, YTS TIGIT cells, BW cells, BW TIGIT cells and P815 cells. All cells were grown in RPMI-1640 media (Sigma- Aldrich) except for HEK293T cells which were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma- Aldrich). All cell line cultures were supplemented with 10% inactivated fetal bovine serum (Sigma-Aldrich), 1 mM sodium pyruvate (Biological Industries), 2 mM glutamine (Biological Industries), nonessential amino acids (Biological Industries), 100 U/ml penicillin (Biological Industries) and 0.1 mg/ml streptomycin (Biological Industries). Unless noted otherwise, all experiments including mammalian cells were conducted in these media.

Microbial strains

The WT Candida species and strains used in this study were Candida albicans SC5314, Candida glabrata BG2, Candida parapsilosis and Candida krusei. C. albicans deletion mutants used were C. albicans als1 Δ/Δ1467 , als2Δ/Δ 2757, als3Δ/Δ 1843, als4Δ/Δ 2034, als5Δ/Δ 2373, als6Δ/Δ 1420, als7 Δ/Δ 1429 and als9Δ/Δ 2028, which are all derivatives of SC5314.

Unless written otherwise, all of the Candida species and strains were kept in -80°C frozen glycerol stocks and grown regularly on Sabouraud dextrose agar plates (Sigma- Aldrich) for up to 4 weeks. Prior to an experiment the fungi were inoculated into Sabouraud dextrose broth (Sigma-Aldrich) and grown overnight in 30°C under shaking and aerobic conditions. The overnight culture was diluted into fresh Sabouraud dextrose broth (1:50) and grown for additional 2-4 hours before its introduction into the experiment.

Mice

Mice used in this study were male C57BL/6 mice aged 6-8 weeks. The mice were test- naive and were group-housed under specific pathogen free (SPF) conditions prior to their use. Littermates were randomly allocated to the different experimental groups. All experiments were done in the SPF unit of the Hebrew University-Hadassah Medical School (Ein-Kerem, Jerusalem) in accordance with the guidelines of the Declaration of Helsinki and the local research ethics committee.

Human Subjects

Details regarding human subjects relevant to this study are provided in Tables 1 and 2 The clinical trial was approved by the Tel Aviv Sourasky Medical Center Institutional Ethics Committee (approval number 0729-16). The need for informed consent was waived given the retrospective observational nature of this study.

I' low cytometry

Mammalian or fungal cells were grown as described above. At the start of the experiment the cells were washed three times in IxPBS. For each wash the conditions were 515G (for mammalian cells) or 3000G (for fungal cells), for 5 minutes in 4°C. Following the washes, the cells were counted using a hemocytometer and divided into U-bottomed 96-well plates to a concentration of 5 or 10x10⁴ cells/well. Each well was incubated in the presence of primary antibodies (0.25ug/well) or Ig-fusion proteins (0.5-5ug/well) diluted in FACS medium (lx PBS, 0.05% Bovine Serum Albumin, 0.05% NaN3) for 1 hour on ice. The negative control protein used for the experiments was NKp46-Dl-Ig. In instances when the primary antibodies were not fluorophore-conjugated the cells were next washed one time with FACS medium, and then stained with 2^nd antibodies (0.75ug/well) for 30-45 minutes on ice. Finally, the cells were washed 2 times in FACS medium, and analyzed using either a FACSCalibur machine (BD Biosciences) or a CytoFlex machine (Beckman-Coulter Fife Sciences) and the FCS Express software (De Novo Software).

Ig fusion protein generation

The extracellular portion of the fusion proteins used was cloned into a mammalian expression vector (pIRESpuro3) containing a mutated Fc domain of human IgGl adjacent to its integration site. The generation of TIGIT-Ig, NKp46-Dl-Ig, NKp44-Ig and PVR-Ig was described previously (Arnon et al. 2001, Eur. J. Immunol. 31, 2680-2689; Glasner et al. 2012, PLoS One 7(5); Stanietsky, N. et al. 2009, Proc. Natl. Acad. Sci. U. S. A. 106, 17858-17863. NT-Als9-1-Ig and NT-Als9-2-Ig were generated by adding the signal peptide of human CD5 to the sequence of the relevant domains of the original proteins, flanking them with EcoRI and BamHI restriction sites and codon-optimizing the sequence for expression in human cells. The sequences were generated synthetically as gBlocks gene fragments (IDT). The expression vector was amplified in chemically competent DH5a Escherichia coli bacteria grown in Luria Broth in 37°C and extracted from the bacteria using AccuPrep Plasmid Mini Extraction Kit (Bioneer Corporation). The vector was then transfected into HEK293T cells using the reagent TransIT-LTl (Mirus Bio) and following a 48-hour recovery the cells underwent selection using Puromycin (5ug/ml). Surviving colonies were grown separately and measured for fusion protein secretion using ELISA assay performed on their growth media using an anti-human-IgG antibody. The clones secreting the highest amount of protein were propagated and eventually transferred into Low Protein BSA-Free medium (LPM, Biological Industries) complemented with 1 mM sodium pyruvate (Biological Industries), 2 mM glutamine (Biological Industries), nonessential amino acids (Biological Industries), 100 U/ml penicillin (Biological Industries) and 0.1 mg/ml streptomycin (Biological Industries). The medium was collected and Ig-fusion proteins were purified using a HiTrap Protein G HP column (Sigma- Aldrich) in a BioCAD High Pressure Perfusion Chromatography Station (PerSeptive Biosystems). The resulting proteins were buffer-exchanged using dialysis bags into IxPBS. Protein quality and purity were examined using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS/PAGE) followed by Coomassie staining of the gel using Imperial™ Protein Stain (ThermoFisher Scientific). Protein quantity was measured using a Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific).

ELISA

All ELISA experiments were performed in high-binding clear F-bottomed 96-well ELISA plates (De-Groot group). For staining C. albicans cells using Ig-fusion proteins 5x10⁵ cells were plated in each well, centrifuged (3000G, 5 minutes, 4°C) and left to bind to the well for 2 hours in a stationary 30°C incubator. The cells were then blocked using PBS-BSA (1% w/v bovine serum albumin diluted in IxPBS) for 2 hours in room temperature and washed 4 times using PBST (IxPBS supplemented with 0.05% Tween 20). Next, the Ig- fusion proteins were diluted in PBS-BSA and added to the wells (2.5ug/well, final volume lOOul/well) and the plates were incubated for 2 hours on ice. The negative control protein used for the experiments was NKp44-Ig. The plates were then washed again 4 times with PBST and a detection antibody (Biotin-SP-AffiniPure Rabbit Anti-Human IgG, Jackson ImmunoResearch) diluted in PBS-BSA was added for 1 hour at room temperature. Next, the plates were washed 6 times with PBST, incubated for 30 minutes with Strep tavidin-HRP (Jackson ImmunoResearch) and washed 6 times using PBST. Finally, the plates were developed using 3, 3 ’,5, 5 ’-tetramethylbenzidine (TMB) substrate (SouthernBiotech) and read at 650nm.

For the deletion library screen a similar method was used but the fungal cells were directly grown in the ELISA plate wells overnight in Sabouraud dextrose broth (Sigma- Aldrich) and in a 30°C stationary incubator. Following the abovementioned protocol and in order to compensate for different growth rates or adherence strengths of the various mutants, the ELISA plates were washed 6 times with PBST and then 100ul XTT/Menandione mix (0.5gr/L and 1uM, respectively, Sigma- Aldrich) was added to each well. The plates were covered in aluminum foil and incubated for 2 hours in a stationary 37°C incubator. Next, the plates were centrifuged (3000G, 4°C, 5 min), and 80ul/well were transferred to a new plate and then read at 490nm. For each well, the 650nm read was normalized to the 490nm read.

For the IL-2 ELISA following the BW assay or the IFNy ELISA following the T cell activation assay a similar protocol to the one described above for Ig-fusion proteins was used, with the following changes: The ELISA plates were initially coated with IxPBS-diluted anti- mouse-IL2 or anti-human IFNy antibodies (Both from BioLegend) for 2 hours in 37°C or overnight in 4°C. Instead of C. albicans cells, medium from the experimental wells was centrifuged (3000G, 5 minutes, 4°C) and the supernatant was added to the plates, and the detection antibody was biotinylated anti-mouse-IL2 or biotinylated anti-human IFNy (both from BioLegend) from different clones than the coating antibody.

Immunofluorescence microscopy

C. albicans yeast cells were grown as described above. Hyphal cells were grown overnight in 37°C with RPML1640 media (Sigma-Aldrich) supplemented with 10% inactivated fetal bovine serum (Sigma-Aldrich), 1 mM sodium pyruvate (Biological Industries), 2 mM glutamine (Biological Industries), nonessential amino acids (Biological Industries), 100 U/ml penicillin (Biological Industries) and 0.1 mg/ml streptomycin (Biological Industries). Each staining contained 1.5x10⁵ cells. The cells were washed 3 times in ice-cold IxPBS (3000G, 4°C, 5 minutes) and then blocked using CAS-Block (ThermoFisher Scientific) for 1.5 hours in 4°C. The cells were washed again in IxPBS and 15ug of Ig-fusion proteins diluted in CAS-Block were added. The negative control protein used for the experiments was NKp46-Dl-Ig. The cells were incubated with the fusion proteins for 2 hours in 4°C and then washed twice in ice-cold IxPBS. A 2^nd antibody (APC-anti-human IgG, Jackson ImmunoResearch) was added for 1 hour in 4°C and then the cells were washed twice in ice-cold IxPBS. The cells were incubated in the presence of FITC (0. Img/ml, diluted in IxPBS) for 25 minutes, washed twice in ice-cold IxPBS and fixated using 4% PFA (Bar- Naor Ltd) overnight. Finally, the cells were mounted on an 8-chamber slide (Bar-Naor) and visualized using an Olympus Fluoview FV 1000 confocal microscope.

Cytotoxicity assay

Fungal and mammalian cells were grown as described above. The cells were washed 3 times in either sterile 1x PBS or RPML1640 and in the following conditions: 515G (for mammalian cells) or 3000G (for fungal cells), for 5 minutes and in 4°C. Following the washes, the cells were counted using a hemocytometer. Mammalian cells were then incubated in the presence of isotype control or TIGIT -blocking antibodies (1ug/10⁵ cells, diluted in RPML 1640-based growth media described above) for 1 hour on ice. Following the blocking stage, the effector mammalian cells were mixed with the target fungal cells in U-shaped 96- well plates (5x10⁴ mammalian cells, 1000 fungal cells) and in a final volume of 200ul RPML 1640-growth media (described above). The cells were co-incubated for 12-14 hours in a stationary 37°C, 5% CO₂ incubator and then serially diluted in IxPBS and plated on Sabouraud dextrose agar plates (Sigma-Aldrich). The plates were incubated in a stationary 30°C incubator and the number of colonies in each plate was measured after 24-48 hours. The % Colony Forming Units (CFU) reduction was calculated by comparing the CFU of each plate to the CFU measured in a control plate in which a culture identical to all other samples but lacking effector mammalian cells was plated.

T cell activation assay

Primary T cells, P815 cells and C. albicans cells were isolated and cultured as described above. P815 cells were irradiated (6000 RAD), washed in RPMI media and incubated for 1 hour on ice with isotype control or anti-human CD3 antibodies (both from BioLegend). Antibody concentration was 0. lug/2.5x10³ cells. In parallel, the T cells were washed in RPMI media and incubated for 1 hour on ice in the presence of either isotype control or anti-human TIGIT antibodies (BioLegend and in-house, respectively). Antibody concentration was lug/2.5x10³ cells. Following the incubation both cell types were mixed in a U-bottomed 96- well plate format (2.5x10³ cells of each type per well). C. albicans cells were washed in sterile IxPBS 3 times (3000G, 4°C, 5 minutes) and added to the relevant wells (2.5x10³ cells/well). The plates were incubated for 12 hours in a 37°C, 5% CO₂ stationary incubator and then frozen in -20°C. At the day of ELISA, the plates were thawed, centrifuged (3000G, 4°C, 5 minutes), and the top media was taken for ELISA analysis using anti-human IFNy antibodies (BioLegend) as described above.

Murine invasive candidiasis model

Male C57BL/6 mice, aged 6-8 weeks, and C. albicans cells, were grown as described above. At the day of experiment the fungal cells were washed 3 times in IxPBS and taken to the animal facility on ice. The mice were then injected with either 5x10⁵/100ul IxPBS/mouse (for experiments measuring fungal burden or survival after T- or NK-cell depletion) or 6.5x10⁵/100ul PBS/mouse (for survival experiments without depletion). Antibodies used for TIGIT blockade (anti-mouse TIGIT clone 1G9, BioXCell), NK cell depletion (anti-mouse NK1.1 PK136, BioXCell) or T cell depletion (anti-mouse CD3 17A2, BioXCell) were diluted in IxPBS for a final volume of 200ul/mouse and injected intraperitoneally. Injection times were once every 48 hours starting one day prior to infection.

For fungal burden experiments, the mice were sacrificed 48 hours post-infection and relevant organs were harvested. The organs were physically homogenized, filtered through a 70um strainers, serially diluted in ice-cold IxPBS and plated on Sabouraud dextrose agar plates (Sigma- Aldrich). The plates were incubated for 48 hours in a stationary 30°C incubator and once colonies were visible, they were counted.

For survival experiments the mice were weighted and clinically evaluated daily and euthanized once their weight was reduced to <80% of their starting weight. Mice that developed unusual clinical symptoms were noted, euthanized, and removed from the study.

Murine thymoma cell line (BW) assay

BW and C. albicans cells were grown as described above. The cells were then washed three times in either sterile lx PBS or RPMI-1640 using the following conditions: 515G (mammalian cells) / 3000G (fungal cells), 5 minutes, 4°C. The washed cells were then counted using a hemocytometer. The fungal cells were incubated in the presence of 50ug/ml of Fluconazol (BioAvenir) for 1 hour on ice and then the fungal and BW cells were mixed in a F-bottomed 96-well plates in a final volume of 200ul RPMI-1640 growth media described above and supplemented with 50 ug/ml of Fluconazol (BioAvenir). Each experimental well contained 5x10⁴ BW cells and 2.5x10⁴ fungal cells. The cells were co-incubated for 48 hours in a stationary 37°C 5% CO₂ incubator. Finally, the experiment plates were frozen in -20°C until the time of IL-2 secretion measurement. At that time the plates were thawed, centrifuged (3000G, 5 minutes, 4°C) and the top media underwent ELISA for IL-2 measurement as described above.

Microscale Thermophoresis (MST)

The microscale thermophoresis experiments were performed on a blue/red Monolith NT.115 machine (NanoTemper) using TIGIT-Ig protein labeled using the Monolith NT™ Protein Labeling Kit RED-NHS (NanoTemper). Capillary scan at 95% LED power was used to determine minimal fluorescence levels. All experiments were performed under 20% MST power for 30 seconds with 95% LED power in standard capillaries at 25°C. Interaction between labeled TIGIT-Ig and non-labeled NT-Als9-2-Ig or PVR-Ig was measured by maintaining constant levels of TIGIT-Ig and mixing it with a series of target proteins diluted in lx PBS in serially decreasing concentrations. Binding was observed by measuring the change in fluorescence in each capillary containing a receptor-ligand mix. All identified interactions were validated to be denaturation-sensitive by adding DTT and SDS to the samples (final concentration 20mM and 2% respectively), heating them (95°C, 5 minutes), and reading them in the machine.

Quantification and statistical analysis Statistical analysis was performed using either Prism 8 (GraphPad) or Excel (Microsoft). All of the relevant statistical data for the experiments including the statistical test used, value of n, definition of significance, etc. can be found in the figure legends or the relevant methods section.

Example 1. TIGIT directly binds C. albicans yeast and hyphae cells

To test how NK cells recognize C. albicans cells, a library of recombinant fusion proteins, containing the extracellular ligand binding domain of known NK receptors fused to the Fc domain of human IgGl, was created. The binding of the various NK receptors to C. albicans yeast cells was analyzed using flow cytometry. Binding was observed between the fusion protein TIGIT-Ig and the yeast cells (Fig.lA, quantified in Fig. 1B). To corroborate this finding, the cells were bound to 96-well plates and EEISA assays with TIGIT-Ig were uses to examine binding (Fig.1C).

To test whether TIGIT binding is C. albicans-specific, three additional Candida species, C. glabrata, C. parapsilosis and C. krusei were reacted with TIGIT-Ig. Similar to C. albicans, all of the chosen species also have pathogenic potential. Of the three examined species, only C. parapsilosis was significantly stained with TIGIT-Ig (Fig. 1D).

C. albicans is a multi-morphic fungus that can differentiate into several cell types. The most notable example are the elongated hyphal cells which are critical for its pathogenicity and invasive potential. As it is technically problematic to use hyphae in flow cytometry and ELISA experiments, confocal microscopy using TIGIT-Ig fusion protein to stain and compare yeast and hyphal cells was applied. In agreement with the above results, yeast cells were stained using with TIGIT-Ig, but not with a negative control fusion protein. Interestingly, TIGIT-Ig binding to C. albicans hyphae was also observed (data not shown).

Thus, TIGIT binds the two major C. albicans morphotypes.

Example 2. C. albicans binding of TIGIT is functional and leads to inhibition of NK and T cells

To test whether TIGIT recognition of C. albicans is functional, and to directly asses the TIGIT- C. albicans interactions, TIGIT is expressed in the NK cell line YTS Eco which does not endogenously express TIGIT (Fig. 7A-7B). The parental cell line (YTS Eco) and the TIGIT expressing cell line (YTS TIGIT) were co-incubated with C. albicans cells, in the presence or absence of a monoclonal anti-human TIGIT -blocking antibody (VSIG9#1). After 12 hours of co-incubation the cells were serially diluted, plated on Sabouraud agar plates, and the resulting fungal colonies were counted on the following day. Percent Colony Forming Units (CFU) reduction was calculated by comparing the resulting colonies to a control experiment performed in the absence of YTS cells.

TIGIT blockade significantly increased the ability of YTS TIGIT cells to eliminate C. albicans cells but had no effect on the parental YTS Eco cells (Fig. 2A), which indicates that C. albicans activation of TIGIT is functional and inhibitory for NK cells.

Next, the validity of the results was further tested by using primary human NK cells. NK cells were isolates from the blood of various healthy human donors, activated, and co- incubated with C. albicans cells in the presence or absence of a TIGIT -blocking antibody. Remarkably, TIGIT blockade significantly increased the elimination of the fungal cells by NK cells (Fig. 2B).

As TIGIT is expressed on both NK and T cells, inhibition of T cells via TIGIT activation by C. albicans was examined. Primary CD4+TIGIT+ T cells from healthy human donors were isolated and cultured under conditions that favor differentiation into TH1 cells. These T cells were then activated using anti-CD3 antibodies bound to Fc receptor expressed on the murine P815 mastocytoma cell line. This leads to TCR crosslinking, T cell activation, and IFNy secretion (Fig. 2C). It was expected that addition of the anti-TIGIT blocking antibody would enhance T cell activity, but only in the presence of a TIGIT- stimulating signal, in this case, C. albicans (Fig. 2C). Indeed, TIGIT blockade in the absence of C. albicans did not lead to increased IFNy secretion, but in the presence of C. albicans a significant elevation in IFNy secretion was observed (Fig. 2D).

Thus, C. albicans recognition by TIGIT is functional in-vitro in both NK and T cells, leading to their inhibition.

Example 3. TIGIT manipulation by C. albicans is an immune-evasion mechanism in- vivo

Next, it was tested whether the C. albicans recognition by TIGIT is functional in-vivo. First, the relevance of T and NK cells was tested. C56BL/6 mice were depleted of either NK cells, T cells or not depleted and then injected intravenously with C. albicans yeast cells. Mouse weight was monitored as the main measure of disease severity. Weight reduction of 20% or more was regarded as the ethical endpoint of the experiment. A significant increase in mortality was observed after depletion of either T or NK cells, and no difference was observed between mice lacking either population (Fig. 3A), leading to the conclusion that both NK and T cells are important in the control of invasive candidiasis.

To test the role of TIGIT during invasive candidiasis, mice were pre-treated with a commercial murine anti-TIGIT blocking antibody and then infected intravenously with C. albicans. The fungal burden in the kidneys, the major focus of C. albicans infection in this model, was determined 48 hours post-infection (Fig. 3B). A significant reduction in fungal burden following TIGIT blockade was observed, in agreement with a role for TIGIT in C. albicans immune evasion (Fig. 3C).

Example 4. TIGIT manipulation by C. albicans affects human clinical disease

While the proof-of-concept experiments showed a significant role for TIGIT manipulation during invasive candidiasis in mice, it was desired to investigate whether it might also have a role in human clinical disease. Clinical isolates of C. albicans were collected from a cohort of 105 human patients who suffered from C. albicans bloodstream infections. The ability of these clinical isolates to manipulate TIGIT was initially determined using the BW assay. The BW assay is a semi-functional screen in which the extracellular portion of human TIGIT is fused to a murine -chain and expressed in the murine thymoma

cell line BW. Functional binding of a ligand to TIGIT leads to activation of the BW cells and secretion of IL-2. This is a very robust and sensitive assay which was even used in patients to determine antibody titers against various viruses. BW-TIGIT cells (Fig. 7C and 7D) were incubated in the presence or absence of fluconazole-inactivated C. albicans cells for 48 hours and IL-2 levels were measured using ELISA. TIGIT activation ability of each clinical strain was measured and compared to the WT lab strain SC5314. Importantly, all clinical strains activated TIGIT, as they all activate BW-TIGIT cells similarly to the WT lab strain (Fig. 4A). Interestingly, five strains with increased TIGIT activation were identified (marked in gray in Fig. 4A).

It was further examined whether the increased ability of the five human isolates to activate TIGIT was clinically relevant. For this, clinical data of these five isolates was blindly compared to the rest of the cohort. It was found that the strains that had increased TIGIT activation capabilities also had a significantly longer time to blood clearance. No significant differences in other clinical parameters including 30-day mortality were observed (Fig. 4B, Tables 1, 2 and 3). The results indicate that the increased ability of these five human isolates to activate TIGIT enables the invading fungal cells to survive longer during infection.

Table 1. Survival and time for blood clearance data from the human invasive candidiasis cohort presented in Figure 4A.

Table 2. Epidemiological data regarding the invasive candidiasis patient cohort

Table 3. The origins of candidemia for the invasive candidiasis patient cohort, p-value for categorical data was calculated using Fischer’s exact test, and for quantitative data the Student’s T test was used.

Example 5. Als proteins of C. albicans are fungal TIGIT ligands

To identify the fungal ligand of TIGIT, a library of 1322 C. albicans deletion mutants, encompassing ~11% of the C. albicans genome was scanned (Noble et al., 2010) and screened in an ELISA assay as described in the Materials and Methods section. The ELISA assay was used for this screen since it enables parallel screening of a large number of fungal strains. However, no mutant was found to alter TIGIT-Ig recognition.

Next, additional C. albicans proteins not represented in the deletion library were tested. Microbial NK ligands such as the Epa proteins of the fungus C. glabrata, the Fap2 protein of the bacterium F. nucleatum, and viral proteins such as the influenza virus protein HA share several characteristics such as being important pathogenicity factors, functioning as adhesion molecules, and are localized to the cell surface of the pathogen or the cells it infects. An important C. albicans protein family that shares all of these characteristics and is almost not present in the deletion library (other than a single member, Als5) is the Agglutinin-Like Sequences (Als) family.

In order to check whether Als proteins could serve as TIGIT ligands, a BW assay using BW-TIGIT cells, was performed with a series of C. albicans mutants, each deleted for both alleles of a member of the Als protein family. This assay for screening was selected because it is a semi functional assay, suitable for screening a small number of mutants. The ability of each strain to activate TIGIT was compared to the WT strain and three Als mutants impaired in TIGIT activation; als6Δ/Δ, als7Δ/Δ and als9Δ/Δ were identified (Fig. 5A). The importance of these genes in TIGIT -mediated immune evasion was then examined using primary human NK cells used in a cytotoxicity assay. Similar to the results presented in Fig. 5A, only the WT strain of C. albicans was sensitive to TIGIT blockade, while the deletion strains als6Δ/Δ, als7Δ/Δ or als9Δ/Δ. were not (Fig. 5B).

To test whether Als9 (ALS9 has two alleles; ALS9-1 and ALS9-2) can directly interact with TIGIT, two fusion proteins containing the N’ -terminal (NT) ligand binding domain of Als9-1 or Als9-2 fused to the Fc domain of human IgGl were generated. The new proteins were named NT-Als9-1-Ig and NT-Als9-2-Ig. In addition, the interaction of Als6 and Als7 with TIGIT was examined. Fusion proteins containing the N’ -terminal (NT) ligand binding domain of Als6 or Als7 fused to the Fc domain of human IgGl were generated. Flow cytometry was used to check whether one of the Als9 fusion proteins directly binds TIGIT by staining parental BW cells (which do not express TIGIT, Fig. 7C-7D) and BW cells expressing the chain-TIGIT. NT-Als9-2-Ig stained the TIGIT-expressing cells

specifically, while NT-Als9-1-Ig did not stain any of the cells (Fig. 5C, quantified in D, and data not shown). The differential binding of TIGIT by the two ALS9 alleles is similar to previous reports of distinct ligands and functions of the two gene copies (Zhao et al. 2007, Microbiology 153, 2342-2350).

Flow cytometry was further used to check whether Als6 and Als7 fusion proteins directly bind TIGIT by staining parental BW cells and BW cells expressing the chain-

TIGIT. As shown in Figures 5C and 5D, NT-Als7-Ig and NT-Als6-Ig stained the TIGIT- expressing cells specifically.

To validate this direct interaction and determine its affinity Microscale Thermophoresis (MST) was used to compare the fusion proteins binding to the binding of TIGIT to one of its human ligands, PVR. A direct, denaturation-sensitive interaction between TIGIT and the fusion proteins was observed with a KD of 455 nM for NT-Als9-2, a KD of 42 nM for NT- Als7-Ig, and a KD of 6 for NT-Als6-Ig. These interactions were at the same order of magnitude as that of TIGIT and PVR (49 nM) (Fig. 5E).

Example 6. Als9, Als7 and Als6 -mediated immune evasion can be targeted using immunotherapy in mice.

Finally, to assess that the Als9, Als7, and Als6 proteins are indeed TIGIT ligands, mice were infected intravenously with als9Δ/Δ, als7Δ/Δ, als6Δ/Δ or the WT strain of C. albicans in the presence or absence of TIGIT blockade. Similar to the results presented in Figure 3C, TIGIT blockade significantly reduced fungal burden during infection with the WT strain. Importantly, TIGIT blockage did not reduce fungal burden when als9Δ/Δ, als7Δ/Δ, or als6Δ/Δ were the infecting agent (Fig.6A).

Lastly, it was checked whether immune checkpoint blockade with aTIGIT antibody has a therapeutic anti-candidal potential. Mice were infected intravenously with either WT, als9Δ/Δ, als7Δ/Δ, or als6Δ/Δ C. albicans, treated with either mock or a TIGIT -blocking antibody, and followed their clinical progress. Remarkably, treatment with an aTIGIT antibody reduced mortality and led to a significant clinical improvement of mice infected with WT C. albicans. However, when the als deletion strains were used, TIGIT blockage had no such effect (Fig. 6B).

In summary, a new interaction between the immune checkpoint receptor TIGIT and C. albicans is shown here. This interaction leads to the inhibition of NK and T cell antifungal activities in both humans and mice, both in-vitro and in-vivo, in disease models and in a cohort of more than 100 human patients. The fungal ligands responsible for the manipulation of TIGIT are members of the Als adhesin proteins: Als9-2, Als6 and Als7. It is now shown that the anti-TIGIT escribed herein, VSIG9#1, can be successfully used for the treatment of C. albicans infection.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

Claims

1. An isolated monoclonal antibody or an antibody fragment thereof comprising at least the antigen binding portion, for use in treating a fungal infection or disease, or a symptom associated with a fungal infection, the antibody is capable of blocking the interaction between human T-cell immunoglobulin and ITIM domain (TIGIT) and a fungal agglutinin-like sequence (ALS) protein selected from the group consisting of Als6, Als7, and Als9.

2. The isolated monoclonal antibody or the antibody fragment for use according to claim 1, wherein the antibody is capable of blocking the interaction between human TIGIT and Als9.

3. The isolated monoclonal antibody or the antibody fragment for use according to claim 1, wherein the isolated antibody or antibody fragment comprises three heavy-chain (HC) complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 7 and three light-chain (LC) CDRs of a light-chain variable region set forth in SEQ ID NO: 8, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence.

4. The isolated monoclonal antibody or the antibody fragment for use according to any one of claims 1 to 3, wherein the fungal infection is caused by a pathogen selected from the group consisting of Candida, Saccharomyces, Pneumocystis, Aspergillus, and Cryptococcus.

5. The isolated monoclonal antibody or the antibody fragment for use according to claim

4, wherein the fungal infection is caused by a Candida species.

6. The isolated monoclonal antibody or the antibody fragment for use according to claim

5, wherein the Candida species is selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida auris, and Candida tropicalis.

7. The isolated monoclonal antibody or the antibody fragment for use according to any one of claim 5 or 6, wherein the fungal infection is candidiasis.

8. The isolated monoclonal antibody or the antibody fragment for use according to claim 7, wherein the candidiasis is mucosal candidiasis.

9. The isolated monoclonal antibody or the antibody fragment for use according to claim 7, wherein the candidiasis is invasive candidiasis.

10. The isolated monoclonal antibody or the antibody fragment for use according to any one of claims 1 to 9, the antibody comprises a set of six CDRs, wherein the HC CDR1 comprises the sequence GYTFTSYGIS (SEQ ID NO: 1); HC CDR2 comprises the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO: 2); and HC CDR3 comprises the sequence: KGPYYTKNEDY (SEQ ID NO: 3).

11. The isolated monoclonal antibody or the antibody fragment for use according to any one of claims 1 to 10, the antibody comprises a set of six CDRs wherein the LC CDR1 comprises the sequence: RASEHIYYSLA (SEQ ID NO: 4); LC CDR2 comprises the sequence: NANSLED (SEQ ID NO: 5); and LC CDR3 comprises the sequence: KQAYDVPRT (SEQ ID NO: 6).

12. The isolated monoclonal antibody or the antibody fragment for use according to ant one of claims 1 to 11, the antibody comprises a set of six CDRs wherein the HC CDR1 comprises the sequence GYTFTSYGIS (SEQ ID NO:1); HC CDR2 comprises the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2); HC CDR3 comprises the sequence: KGPYYTKNEDY (SEQ ID NOG); LC CDR1 comprises the sequence: RASEHIYYSLA (SEQ ID NO:4); LC CDR2 comprises the sequence: NANSLED (SEQ ID NOG); and LC CDR3 comprises the sequence: KQAYDVPRT (SEQ ID NO: 6).

13. The isolated monoclonal antibody or the antibody fragment for use according to any one of claims 1 to 12, comprising a heavy chain variable region of SEQ ID NO:7, or an analog having at least 95% sequence similarity with said heavy chain variable region sequence.

14. The isolated monoclonal antibody or the antibody fragment for use according to any one of claims 1 to 13, comprising a light chain variable region of SEQ ID NOG, or an analog having at least 95% sequence similarity with said light chain variable region sequence.

15. The isolated monoclonal antibody or the antibody fragment according to any one of claims 1 to 14, comprising a heavy chain and a light chain, wherein the heavy chain comprises SEQ ID NO:7 and the light chain comprises SEQ ID NOG.

16. A variant of an isolated monoclonal antibody or antibody fragment according to claim

15, having at least 95% identity with said antibody light or heavy chain.

17. The monoclonal antibody or the antibody fragment according to any one of claims 1 to

16, wherein the antibody or antibody fragment binds to an epitope within the human TIGIT protein to which a monoclonal mouse antibody having a heavy chain comprising the amino acid sequence SEQ ID NO:7 and a light chain comprising the amino acid sequence SEQ ID NO:8, binds.

18. The isolated monoclonal antibody of any one of claims 1 to 17, wherein the antibody is selected from the group consisting of: a humanized antibody, a bispecific antibody an antibody conjugate, or a fragment comprising at least the antibody binding portion.

19. A pharmaceutical composition for use in treating or preventing a fungal infection or disease or a symptom associated with a fungal infection, wherein the pharmaceutical composition comprises an isolated antibody or antibody fragment comprising three heavy-chain (HC) complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 7 and three light-chain (LC) CDRs of a light- chain variable region set forth in SEQ ID NO: 8, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence, and an acceptable excipient, diluent, salt or carrier.

20. The pharmaceutical composition for use according to claim 19, wherein the isolated monoclonal antibody or the antibody fragment comprising: HC CDR1 comprises the sequence GYTFTSYGIS (SEQ ID NO:1); HC CDR2 comprises the sequence: EIYPRSGNTYYNEKFKG (SEQ ID NO:2); HC CDR3 comprises the sequence: KGPYYTKNEDY (SEQ ID NOG); LC CDR1 comprises the sequence: RASEHIYYSLA (SEQ ID NO:4); LC CDR2 comprises the sequence: NANSLED (SEQ ID NOG); and LC CDR3 comprises the sequence: KQAYDVPRT (SEQ ID NO: 6).

21. The pharmaceutical composition for use according to any one of claims 19-20, wherein the fungal infection is caused by a Candida species.

22. The pharmaceutical composition for use according to any one of claims 19-21, further comprising an additional anti-fungal agent.

23. A method of treating fungal infection comprising administering to a subject in need thereof, a pharmaceutical composition comprising an effective amount of at least one antibody, antibody fragment or conjugate thereof comprising at least the antigen binding portion, wherein the isolated antibody or antibody fragment comprising three heavy-chain (HC) complementarity determining regions (CDRs) of a heavy-chain variable region set forth in SEQ ID NO: 7 and three light-chain (LC) CDRs of a light- chain variable region set forth in SEQ ID NO: 8, or an analog or derivative thereof having at least 90% sequence identity with said antibody or fragment sequence. The method of claim 23, wherein the fungal infection is caused by a pathogen selected from the group consisting of Candida, Saccharomyces, Pneumocystis, Aspergillus, and Cryptococcus. The method of claim 24, wherein the fungal infection is caused by Candida. The method of claim 25, wherein the fungal infection is candidiasis. The method of claim 26, wherein the candidiasis is caused by a Candida species selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida auris, and Candida tropicalis. The method of claim 26, wherein the Candida infection is caused by a C. albicans infection. The method of claim 26 wherein the candidiasis is mucosal candidiasis. The method of claim 26 wherein the candidiasis is invasive candidiasis. The method of any one of claims 23 to 30, wherein the method further comprising administering or performing at least one additional anti-fungal therapy. The method of claim 31, wherein the anti-fungal agent is selected from the group consisting of Fluconazole, Flucytosine, Nystatin, Amphotericin B, Caspofungin, Voriconazole, natamycin, Posaconazole, lactoferrin, Itraconazole, miconazole, econazole, Micafungin, Ketoconazole, Anidulafungin and Terbinafine.