Antibodies that block HIV1 capsid entry into an FG phase, targeting to and passage through nuclear pore complexes

Abstract

The present invention pertains to the fields of antibody technology, biochemistry, medicine, pharmacology, infection biology, and anti-viral therapy. More specifically, it discloses antibodies, particularly single-domain antibodies, e.g., VHH antibodies that bind an assembled HIV-1 capsid such that the capsid is prevented from entering the permeability barrier of nuclear pore complexes, also called the FG phase. Through the effects of these antibodies, HIV-1 is effectively prevented from entering the cell's nucleus and thus blocked in its life cycle.

Description

[0001] Antibodies that block HIV1 capsid entry into an FG phase, targeting to and passage through nuclear pore complexes

[0002] Field of the invention

[0003] The present invention pertains to the fields of antibody technology, biochemistry, medicine, pharmacology, infection biology, and antiviral therapy. More specifically, it discloses antibodies, particularly single-domain antibodies, e.g., VHH antibodies (VHHs), which bind an assembled HIV-1 capsid such that the capsid is prevented from entering the permeability barrier of nuclear pore complexes, also called the FG phase, and effectively prevent HIV-1 from reaching the cell nucleus. In particular embodiments, the VHHs enforce a premature disintegration of capsids and expose the enclosed genetic material of HIV-1 to cytoplasmic antiviral defense, thereby blocking its life cycle.

[0004] Background of the invention

[0005] HIV-1 (Human immunodeficiency virus 1 ) is the etiologic agent of the Acquired Immune Deficiency Syndrome or AIDS for short, which has caused 40 million deaths to date. Currently ~1.5 million people become newly infected per year. HIV-1 primarily infects and eliminates CD4+ cells (T-helper cells), which are key for humoral immunity. Elimination of CD4+ cells causes an immune defect and prevents the immune system from clearing the infection.

[0006] HIV-1 is a retrovirus. Its genomic RNA is reverse transcribed into DNA after cell entry and then integrated into the host cell genome. Transcription of the HIV-1 locus then not only allows for the production of viral proteins but also of genomic RNA that is packaged in the next generation of viral particles.

[0007] Despite decades of intensive research, no effective vaccine against HIV-1 is available. Anti-retroviral therapy (ART) has greatly advanced but remains difficult because of the high mutation rate of the virus and the latent viral reservoir in CD4+ cells (Menendez- Arias and Delgado, 2022). ART requires a combination of inhibitors of the viral protease, reverse transcriptase, integrase, and cell entry. Still, it is not sufficient for the eradication of the viral reservoir. Thus, such treatment must be continued for life. It is just a management of the infection and not a cure.

[0008] A few cases of a cure have been reported by eliminating immune cells and transplanting hemopoietic stem cells from a healthy donor carrying HIV resistance mutations (see, e.g., Jensen et al., 2023). The extreme side effects make it unlikely that this will become a standard therapy.

[0009] To establish infection, retroviruses must integrate the DNA copy of their reverse transcribed RNA genomes into host chromosomes. The nuclear envelope (NE) is thereby a barrier to overcome. Indeed, most retroviruses rely on mitotic NE breakdown for nuclear entry and therefore only infect proliferating cells. Lentiviruses, such as HIV- 1 , are exceptional in their ability to infect non-dividing cells with intact NEs. Passage through nuclear pore complexes (NPCs) is thus a critical event in the HIV-1 life cycle. NPCs have a mass of ~100 MDa and provide a channel for nucleocytoplasmic transport (Knockenhauer and Schwartz, 2016; Hampoelz etal., 2019) that is controlled by a permeability barrier (Schmidt and Gorlich, 2016). Nuclear transport receptors (NTRs) enable active transport and circulate between the nucleus and cytoplasm. Members of the Importin [3 superfamily represent the largest NTR class. They draw energy from the RanGTPase system, bind cargoes in a RanGTP-controlled manner and translocate them through the NPC barrier. NPCs are built from ~30 different nucleoporins or Nups for short. These include ~10 Nups that anchor barrier-forming phenylalanine-glycine (FG) repeat domains to the NPC scaffold, therefore referred to as FG Nups.

[0010] FG domains have low sequence complexity, are intrinsically disordered, and harbor numerous FG dipeptide motifs that bind NTRs during facilitated translocation. FG motifs can also confer multivalent cohesive interactions, reversibly crosslinking FG domains to a sieve-like FG phase that makes up the permeability barrier (Frey et al., 2006; Frey and Gorlich, 2007; Schmidt and Gorlich, 2015; Lemke, 2016). NTRs and NTR cargo complexes “melt” through such a phase by binding and competing inter- FG-repeat interactions, while inert macromolecules are rejected unless they are recognized as valid cargo.

[0011] The FG domains of Nup98 and its homologs (Wente et al., 1992; Powers et al., 1997) are special in that they occur in very high copy numbers and contribute the largest share of FG mass per NPC (Ori et al., 2013). They feature the highest number (~50) and density (~one per 12 residues) of FG motifs per domain. Water is a poor solvent for Nup98 FG domains; therefore, they readily phase-separate from dilute aqueous solutions, forming a very protein-dense (~400 mg / ml) FG phase (Schmidt and Gorlich, 2015; Ng et al., 2023). This allows the reconstitution of an NPC-like barrier and the study of its properties. Reconstituted Nup98 FG phases recapitulate nuclear transport selectivity indeed very well, fully excluding inert macromolecules such as GFP or mCherry while allowing entry of NTRs and their cargo complexes to very high partition coefficients.

[0012] Early steps of HIV-1 infection, namely surface receptor binding and membrane fusion, ensure the delivery of the viral capsid to the cytoplasm of the target cell (Zila et al., 2021 b). The capsid is composed of ~1500 capsid protein (CA) molecules, arranged in ~250 hexameric and twelve pentameric capsomers (Ganser-Pornillos et al. , 2008). The capsid encloses two genomic RNA copies (chaperoned by the NC protein) as well as two initially required viral enzymes, reverse transcriptase, and integrase (Sundquist and Krausslich, 2012). It was long assumed that the capsid uncoats in the cytoplasm. One argument was that earlier structural models of the NPC had a central channel width of only 40 nm (Pante and Kann, 2002; von Appen et al., 2015) - too narrow to accommodate an intact cone-shaped HIV-1 capsid of 60 x 120 nm. This concept changed recently with studies indicating capsid uncoating inside nuclei (Burdick et al., 2020; Li et al., 2021 ), with new in situ NPC structures showing a ~60 nm wide channel (Schuller et al., 2021 ; Mosalaganti et al., 2022), and electron tomographic reconstructions of an HIV-1 capsid trapped in the central NPC channel (Zila et al., 2021a). However, the capsid must not only fit through the NPC scaffold but also cross the permeability barrier. If it were carried by NTRs like conventional cargo, the additional NTR layer would increase the capsid’s effective diameter, making the passage through the constrained NPC scaffold even less probable. This problem is well illustrated by the fact that the HIV-1 capsid alone is way larger than the experimentally determined ~36 nm size limit for NTR-mediated cargo transport (Pante and Kann, 2002).

[0013] As a solution to this conundrum, the laboratories of the inventors discovered that the HIV-1 capsid behaves itself like an NTR and partitions without the help of trans-acting factors into an otherwise very strict Nup98 FG phase. The inventors now reasoned that this capsid-FG phase interaction might be a suitable target for antiviral intervention, and specifically that antibodies directed against an intact capsid might be able to antagonize the FG phase-entry of HIV-1 capsid species. Expression of such antibodies in human cells should prevent viral entry into nuclei and thus make the expressing cells virus-resistant.

[0014] Summary of the invention

[0015] The present disclosure provides an antibody specifically targeting a lentiviral capsid. The antibody inhibits partitioning of the capsid into an assembled FG phase and can inhibit targeting of the lentiviral capsid to nuclear pore complexes. Mechanistically, these antibodies likely inhibit FG-phase entry either by direct competition with the FG- binding site on CA, or by destabilizing the capsid assembly, or both. The antibody and a nucleic acid molecule encoding said antibody are suitable for diagnostic, prophylactic and / or therapeutic applications.

[0016] In certain embodiments, the lentiviral capsid is a human immunodeficiency virus (HIV) capsid, e.g., a HIV-1 or HIV-2 capsid.

[0017] In certain embodiments, the antibody is a single chain antibody, a single domain antibody or a VHH antibody. In certain embodiments, the antibody is in monovalent form, e.g., as a single domain VHH antibody. The antibody may be fused to a heterologous moiety, e.g., a heterologous protein such as an antiviral effector protein or a different VHH antibody that recognizes a non-overlapping epitope, e.g., as bivalent tandem fusion. In certain embodiments, the VHH antibody may be in multivalent form, to possibly enhance beneficial effects of the fused moieties. The use in a monovalent format is possible due to the very high affinity of the antibody. In certain embodiments, the single domain antibody has - in the monovalent format - a target affinity in the picomolar range.

[0018] A first aspect of the present disclosure is an antibody specifically targeting a lentiviral capsid, e.g., a HIV-1 capsid, as described herein. Preferred VHH antibodies of the present invention, their designations, binding characteristics, i.e., as well as their thermostabilities are listed in the following Table 1 :

[0019] Table 1: Properties of disclosed anti-HIV-1 capsid VHH antibodies. For details see Examples, Figures, and main text. Quantitation of the inhibitory effects on capsid partitioning into the FG phase was as in Figures 4 but based on a larger dataset. VHHs marked by * cause a disassembly of CLPs (capsid-like particles based on wildtype CA). n.d. = not determined.

[0020] A non-limitative list of VHH antibodies of the present invention and their CDR sequences is shown in the following Table 2:

[0021] Table 2: VHH antibodies, their SEQ. IDs and CDRs

[0022] A list of the complete amino acid sequences of the VHH antibodies is shown at the end of the specification.

[0023] A further aspect of the present invention relates to a set of two or more different antibodies directed against a lentiviral antigen, wherein at least one antibody is specifically targeting a lentiviral capsid, e.g., a VHH antibody as described above. In certain embodiments, the set comprises two or more different antibodies specifically targeting a lentiviral capsid each belonging to a different class as shown in Table 1 .

[0024] An antibody described above may be used in medicine, e.g., human medicine, particularly for use in therapy, e.g., in the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with a lentivirus, e.g., an infection with HIV-1 , or in diagnostics, e.g., for detecting HIV-1 in a patient sample, e.g., in a body fluid or tissue sample, or in research.

[0025] Still a further aspect of the invention relates to a nucleic acid molecule encoding an antibody as described above, particularly in operative linkage with a heterologous expression control sequence, a vector comprising said nucleic acid molecule or a recombinant cell or non-human organism transformed or transfected with said nucleic acid molecule or said vector.

[0026] A therapeutic aspect of the invention is based on the direct production of the antibody in the cell to be protected from a virus infection within a subject in need thereof. In certain embodiments, a nucleic acid molecule that encodes the antibody or a vector comprising said nucleic acid molecule is introduced into a cell to be protected and integrated in its genome. Expression of the antibody within the cytoplasm provides immunity against an infection with a lentivirus, e.g., an infection with HIV-1.

[0027] In certain embodiments, the nucleic acid molecule or the vector is introduced into a CD4+ lymphocyte or a precursor cell thereof, e.g., a hematopoietic stem cell.

[0028] In certain embodiments, the cell is obtained from the subject in need of being protected from a virus infection and re-introduced into the subject after being transformed or transfected with the nucleic acid molecule or the vector. In particular embodiments, the subject is a human subject.

[0029] The diagnostic concept of the invention is based the detection of a lentivirus, e.g., HIV- 1 , in a sample, e.g., in a blood, serum or plasma sample. For these purposes, the antibody may be recombinantly produced by known methods.

[0030] Still a further aspect of the invention relates to a method for the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with a lentivirus, particularly by an infection with HIV-1 , comprising administering an effective dose of an antibody or a nucleic acid molecule or a vector encoding the antibody as described above or a recombinant cell transformed or transfected with the nucleic acid molecule or the vector to a subject in need thereof, particularly to a human subject.

[0031] In certain embodiments, a nucleic acid molecule or a vector encoding the antibody as described above is administered to a cell obtained from a subject, particularly to a cell obtained from a human subject.

[0032] Embodiments of the invention

[0033] In the following, specific embodiments of the invention are disclosed as follows:

[0034] 1. An antibody specifically targeting a lentiviral capsid wherein said antibody inhibits partitioning of said capsid into an assembled FG phase. The antibody of embodiment 1 which inhibits partitioning of said capsid into an assembled FG phase by competing with FG-binding site of the assembled capsid, by providing FG-phobic (FG repellent) moieties, and / or by disintegrating the capsid. The antibody of embodiment 1 or 2 which reduces partitioning of a lentiviral capsid, particularly a capsid-like particle, into an in vitro assembled FG phase to 5% residual FG partitioning or less, particularly to 2% residual FG partitioning or less, and more particularly to 1 % residual FG partitioning or less when measured inside an FG particle. The antibody of any one of the preceding embodiments, wherein the binding affinity to a lentiviral capsid, particularly a CA protein-hexamer and more particularly a capsid-like particle, expressed as dissociation constant KD is about 5 nM or less, about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less. The antibody of any one of the preceding embodiments which inhibits targeting of a lentiviral capsid to a nuclear pore complex. The antibody of any one of the preceding embodiments which causes destabilization and / or disintegration of a lentiviral capsid. The antibody of any one of the preceding embodiments which is a single chain antibody. The antibody of any one of the preceding embodiments which is a single domain antibody. The antibody of any one of the preceding embodiments which is a VHH antibody. The antibody of any one of the preceding embodiments wherein the lentivirus is an HIV-1. The antibody of any one of the preceding embodiments which is covalently or non-covalently coupled to a heterologous moiety, particularly a heterologous polypeptide moiety. The antibody of embodiment 10 wherein the heterologous moiety is another VHH antibody. The antibody of embodiment 10 wherein the heterologous moiety is an effector polypeptide, particularly a negatively charged peptide moiety or an antiviral effector. The antibody of embodiment 10 wherein the heterologous moiety is a label. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody Re39E04 having a VHH sequence as shown in SEQ. ID NO: 19 for the binding to a HIV-1 capsid. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody Re39B08 having a VHH sequence as shown in SEQ. ID NO: 31 for the binding to a HIV-1 capsid. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody Re39G05 having a VHH sequence as shown in SEQ. ID NO: 35 for the binding to a HIV-1 capsid. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody Re39B09 having a VHH sequence as shown in SEQ. ID NO: 39 for the binding to a HIV-1 capsid. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody Re56H05 having a VHH sequence as shown in SEQ. ID NO: 43 for the binding to a HIV-1 capsid. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody Re56B09 having a VHH sequence as shown in SEQ. ID NO: 47 for the binding to a HIV-1 capsid. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody Re39G12 having a VHH sequence as shown in SEQ. ID NO: 51 for the binding to a HIV-1 capsid. The antibody of any one of embodiments 1 -13 which competes with the VHH antibody KG11 D11 having a VHH sequence as shown in SEQ. ID NO: 55 for the binding to a HIV-1 capsid. The antibody of any of the preceding embodiments, comprising

[0035] (a) a CDR3 sequence as shown in SEQ. ID NO: 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58 or 62; or

[0036] (b) a CDR3 sequence, which has an identity of at least 80%, at least 90% or at least 95% to a CDR3 sequence of (a). The antibody of any one of the preceding embodiments, comprising

[0037] (a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 16-18, 20-22, 24-26, 28-30, 32-34, 36-38, 40-42, 44-46, 48-50, 52-54, 56- 58 or 60-62; or

[0038] (b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a). The antibody of any one of the preceding embodiments, comprising

[0039] (a) a VHH sequence as shown in SEQ. ID NO: 15, 19, 23, 27, 31 , 35, 39, 43, 47, 51 , 55 or 59; (b) a VHH sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a), particularly a humanized VHH variant of the VHH sequence of (a) which differs from the original VHH sequence by at least one framework mutation, e.g., by 1 to 10 framework mutations, whereby the identity to a human immunoglobulin variable region, e.g., a human germline immunoglobulin variable region is increased. The antibody of any one of the preceding embodiments which is stable, particularly thermostable, or hyperthermostable. The antibody of embodiment 26 which has a melting temperature of at least about 40°C, of at least about 50°C, of at least about 60°C, of at least 80°C or of at least about 95°C when measured under reducing conditions. The antibody of embodiment 26 or 27 which has a melting temperature of at least about 60°C, of at least about 80°C, of at least about 90°C, or of at least about 95°C when measured under non-reducing conditions. The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re39A08 having a VHH sequence as shown in SEQ. ID NO: 15 or a VHH antibody, which is a variant thereof including a humanized variant. The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re39E04 having a VHH sequence as shown in SEQ. ID NO: 19 or a VHH antibody, which is a variant thereof including a humanized variant . The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re39H08 having a VHH sequence as shown in SEQ. ID NO : 23 or a VHH antibody, which is a variant thereof including a humanized variant. The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re56F11 having a VHH sequence as shown in SEQ. ID NO: 27 or a VHH antibody, which is a variant thereof including a humanized variant . The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re39B08 having a VHH sequence as shown in SEQ. ID NO: 31 or a VHH antibody, which is a variant thereof including a humanized variant. The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re39G05 having a VHH sequence as shown in SEQ. ID NO: 35 or a VHH antibody, which is a variant thereof including a humanized variant . The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re39B09 having a VHH sequence as shown in SEQ. ID NO: 39 or a VHH antibody, which is a variant thereof including a humanized variant. The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re56H05 having a VHH sequence as shown in SEQ. ID NO: 43 or a VHH antibody, which is a variant thereof including a humanized variant . The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re56B09 having a VHH sequence as shown in SEQ. ID NO: 47 or a VHH antibody, which is a variant thereof including a humanized variant . The antibody of any one of embodiments 1 -28, which is selected from VHH antibody Re39G12 having a VHH sequence as shown in SEQ. ID NO: 51 or a VHH antibody, which is a variant thereof including a humanized variant. The antibody of any one of embodiments 1 -28, which is selected from VHH antibody KG11 D11 having a VHH sequence as shown in SEQ. ID NO: 55 or a VHH antibody, which is a variant thereof including a humanized variant. The antibody of any one of embodiments 1 -28, which is selected from VHH antibody KG12D02 having a VHH sequence as shown in SEQ. ID NO: 51 or a VHH antibody, which is a variant thereof including a humanized variant. The antibody of any one of the preceding embodiments, which is nonglycosylated, or which is glycosylated. The antibody of any one of the preceding embodiments, which is produced in a bacterium, e.g., E. coli, or in a yeast, e.g., Pichia pastoris, or in a human or animal cell, e.g., an insect cell or a mammalian cell. The antibody of any one of the preceding embodiments, which is in a monovalent format. The antibody of any one of embodiments 1 -42, which is in a multimeric format. The antibody of embodiment 44, which is in a dimeric format. The antibody of embodiment 45, which is in a homodimeric or in a heterodimeric format. A set of two or more different antibodies directed against a lentivirus antigen, particularly a HIV-1 antigen, wherein at least one of said antibodies targets a lentiviral capsid and inhibits partitioning of said capsid into an assembled FG phase. The set of embodiment 47 comprising at least one antibody of any of embodiments 14-25 or 29-40, particularly at least one VHH antibody in a monovalent format. A nucleic acid molecule encoding an antibody according to any one of embodiments 1 -46, preferably in operative linkage with a heterologous expression control sequence. A vector comprising a nucleic acid molecule according to embodiment 49. The vector of embodiment 50, which is suitable for integration into the genome of a host cell, e.g., by homologous recombination, transposon-mediated integration or lentiviral integration. A recombinant cell or non-human organism transformed or transfected with a nucleic acid molecule according to embodiment 49 or a vector according to embodiment 50 or 51 . The recombinant cell of embodiment 52 which is a human cell. The recombinant cell of any one of embodiments 52-53 which is a lymphocyte, particularly a CD4+ lymphocyte or a precursor cell thereof, more particularly wherein the precursor cell is a hematopoietic stem cell. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for use in therapy. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for use in the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with a lentivirus. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of embodiment 49 or 50 wherein the lentivirus is HIV-1 . The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 49-51 in a human subject. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 49-52, wherein the antibody is directedly produced in a cell to be protected. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 55-59, wherein the nucleic acid molecule or the vector is introduced into a cell to be protected and integrated in its genome and wherein the antibody is expressed within the cytoplasm thereby providing immunity against an infection with a lentivirus. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 55-60, wherein a cell is obtained from a subject in need of being protected from a virus infection and re-introduced into the subject after being transformed or transfected with the nucleic acid molecule or the vector. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 55-61 as a monotherapy. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 55-61 as a combination therapy with at least one further therapy. The antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 55-61 or 63 as a combination therapy with an anti-retroviral agent. A pharmaceutical composition comprising as an active agent the antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 for the use of any one of embodiments 55-64 and a pharmaceutically acceptable carrier. A method for the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with a lentivirus comprising administering an effective dose of the antibody of any one of embodiments 1 -46, the set of embodiment 47 or 48, the nucleic acid molecule of embodiment 49, the vector of embodiment 50 or 51 or the cell of any one of embodiments 52-54 or the pharmaceutical composition of embodiment 65 to a subject in need thereof, particularly to a human subject. Use of the antibody of any one of embodiments 1 -46 or the set of embodiment 47 or 48 for detecting a lentivirus, particularly HIV-1 in a sample. The use of embodiment 67, wherein the sample is a biological sample, e.g., a blood, serum or plasma sample. Description of the invention

[0040] The inventors reconstituted three different lentiviral capsid species in vitro, each derived from the HIV-1 CA protein. The first one were ‘40 nm capsid spheres’ assembled from a CA-N21 C / A22C double mutant (Lau et al., 2020) and labeled at a 1 :6 molar ratio with a GFP tracer fused to the C terminus of CA and pointing to the capsid’s interior. The GFP-label was introduced to allow detection by confocal laser scanning microscopy. Negative stain electron microscopy was used to validate the successful assembly (Figure 1 D). The 40 nm capsid spheres are very robust but smaller than the authentic capsid.

[0041] Therefore, also capsid-like particles (CLPs) were assembled from wild-type CA (Schirra et al., 2023). These form assemblies in the size range of the authentic HIV-1 capsid (~60 x 120 nm), including the typical cone shape. In one variant, they were labelled again with a covalently attached GFP-tracer. In the other, mCherry was non- covalently enclosed into the capsid’s interior. The assembly procedures are detailed in Examples 1 -2. Figure 1 documents the capsid assembly by size exclusion chromatography and electron microscopy.

[0042] To study the entry of HIV-1 capsids into the permeability barrier of nuclear pore complexes (NPCs), the inventors employed a defined experimental system based on a well-characterized, perfectly repeated FG repeat domain (Ng etal., 2021 , 2023). This domain is derived from the MacNup98 FG domain of T. thermophila (Schmidt and Gorlich, 2015) and comprises 52 perfectly repeated GLFG peptides of the sequence GGLFGGNTQPAT.

[0043] The FG repeat domain is initially dissolved at a 1 mM concentration in 2 M guanidinium hydrochloride to initially suppress cohesive interactions. After a 100-fold dilution in guanidinium-free buffer, it phase-separates to form a very protein-dense (~400- 500 mg / ml) FG phase - visible in bright field microscopy or confocal laser scans as pm-sized spherical objects (Figure 2). This phase excludes the 25 kDa sized mCherry protein with a partition coefficient of < 0.05 (defined by the ratio of 'intra-phase': 'surrounding buffer' signals). It also excludes a fusion of GFP to the IBB domain (the importin [3-binding domain of importin a) - a very strong nuclear import signal that stays silent in the absence of the cognate importin (Gdrlich et al., 1996). When, however, importin [3 was also added, then the partition coefficient of the IBB-GFP fusion increased from 0.2 to 100, whereby the signal-less mCherry remained excluded as before (Figure 2A). These controls validate that the reconstituted FG phase behaves like the permeability barrier of NPCs.

[0044] Remarkably, the 40 nm capsid spheres, the covalently GFP-labelled CLPs as well as the CLPs filled non-covalently with the mCherry tracer all entered the FG phase and reached partition coefficients of up to 500 (Figure 2B-D, Example 4). This high partitioning occurred without having to add any trans-acting factor (such an importin). Thus, all three capsid species behave like an NTR. The control with the non-covalently capsid enclosed mCherry confirmed that the capsid species remained intact when entering the phase and illustrates that the capsid can serve as a cargo container. The capsid is normally filled with HIV genomic RNA and thus presumably delivers this viral genetic information through the FG phase of NPCs into the nucleus.

[0045] An important detail is that an efficient partitioning into the FG phase required a proper capsid assembly. Partially assembled capsids, such as the mainly hexameric capsomers, only bound to the surface of the FG phase and showed an ~ 200-fold lower partition coefficient than the 40 nm capsid spheres (see Figure 2C).

[0046] VHH antibodies, also called nanobodies, are single domain antibodies corresponding to the antigen-binding domain of camelid heavy-chain only antibodies (Hamers- Casterman et al., 1993; Arbabi Ghahroudi et al., 1997). To obtain anti-capsid VHHs, the inventors immunized an Alpaca with several CA assemblies, namely disulfide- stabilized CA hexamers, capsid spheres, and CLPs. They prepared an immune library and performed phage display using a biotinylated CA hexamer as bait. This retrieved twelve VHH antibodies as specific hexamer binders (see Table 1 for an overview). These VHH antibodies belong to 8 classes (Figure 3 and Table 1 - 2). They feature affinities for the CA hexamer in the low nanomolar to picomolar range (Table 1 , Example 5). The selected VHH antibodies include mostly hyperthermostable ones, and even several that resist unfolding at 95°C even when produced under reducing conditions, i.e., when the structural disulfide bond had not formed (Table 1 ). This is a desired property, considering that the VHHs may be expressed in the reducing cytosol of human cells, where they should stably fold and show only low turnover.

[0047] In a next step, the inventors asked how the anti-capsid VHHs affect the capsid partitioning into the FG phase. Indeed, some of the tested VHH antibodies, such as Re39A08, Re39E04, Re39G05, Re56F11 , and Re56H05, had a very striking effect of essentially abolishing the FG phase entry of the 40 nm capsid spheres (Figure 4, and Table 1 ). There were differences though. The phase entry inhibition was greatest for Re39A08 and Re39E04 (leaving less than 1 % of the capsid signal inside the FG particles), while Re39B09 and Re39G05 still left a rather prominent capsid signal at the phase surface. This difference illustrates that the inhibition of phase entry is not a trivial effect. As seen in Figures 5 and 6, the VHH-imposed block of FG phase entry of the two CLPs species was complete with all four tested VHHs (Re39A08, Re39B09, Re39E04, Re39G05). This difference to the 40 nm capsid spheres can be explained, e.g., by the larger size of the CLPs and by subtle differences in capsomer geometries (capsid spheres have a higher content of CA pentamers), and by differences in stability. While 40 nm capsid spheres are stabilized by engineered disulfide bridges, CLPs are not. CLPs can therefore disassemble when the assembly equilibrium gets shifted.

[0048] There are several ways to explain how VHH antibodies block FG-phase entry of lentiviral capsids. First, VHH antibodies might mask the FG interaction sites of the capsid. Different VHH antibodies may shield these sites more or less efficiently, resulting in varying degrees of FG phase entry inhibition. Given that different members of the same class (likely to have identical epitopes), with just a few exchanged residues, do vary in their effects on FG phase entry, it appears likely that exposed, non- paratopic residues of the VHHs also matter and confer an FG phase repulsion. This would represent a second inhibitory mechanism. Exploiting this consideration and fusing an FG-repellent, negatively charged amino acid sequence (Frey et al., 2018) to the VHHs indeed amplified their inhibitory effect on capsid entry into the FG phase (Figure 7). This was particularly obvious with more sensitive confocal scan settings. The FG phase tested comprised only a single type of FG repeat. Human NPCs, however, contain 11 different FG nucleoporins with rather heterogeneous FG repeat domains. It was therefore critical to also test the effects VHHs on capsid-targeting to authentic NPCs. Therefore, the inventors used digitonin-semipermeabilized HeLa cells (Adam, 1990; Ribbeck and Gorlich, 2001 ) as a complementary experimental system. The digitonin treatment perforates the plasma membranes allowing an entry of transport substrates into the cells. Indeed, when GFP-labelled capsid spheres were added, they accumulated brightly at the NPCs of these cells, colocalizing with an anti- Nup133 VHH antibody that served as an NPC marker (Figure 8). Strikingly this NPC- targeting was greatly suppressed by anti-capsid VHH antibodies fused to the FG- phobic, negatively charged tail (Figure 8).

[0049] A third mechanism of action may involve destabilization and / or disintegration of the capsid by a VHH antibody. As the inventors show in Figure 2c, partially assembled CA capsomers hardly enter the FG-phase on their own but require the assembly into a capsid or CLP. VHH antibodies that shift the assembly equilibrium away from the fully assembled state should therefore also impede the entry in the FG phase. This might happen by VHH antibodies that stabilize a partially assembled or an un-assembled state of the CA protein. Of the here disclosed VHH antibodies, four classes have such an effect. These are represented by Re39G05, Re39B09, Re39G12, and KG11 D11. The effect of these VHHs can be documented by size-exclusion chromatography, where the CLP peak disappears when the capsids are incubated with such VHH antibodies or by negative-stain electron microscopy (EM) as shown in Figure 9: Electron micrographs with capsid-stabilizing VHH (e.g. Re39A08, Re39E04, Re39H08) show CLPs with an additional VHH coat, while CLP structures disappeared after treatment with the capsid-disintegrating VHHs Re39G05, Re39B09, Re39G12, or KG11 D11.

[0050] These CLP-disintegrating VHH antibodies also prevent an entry of GFP-labelled CLPs into the FG phase (Figure 10). While a weak surface signal remained in the presence of Re39G12, perhaps because of its relatively low affinity (Table 1 ), CLP entry into the FG phase was completely blocked by Re39G05, Re39B09, or KG11 D11. The magnitude of the inhibitory effect is consistent with capsid decay. If such VHH- mediated capsid disintegration were to occur in the cytoplasm of a target cell, not only would capsid-mediated import of the viral genome into the nucleus be blocked, but also the capsid content would be released and presented to antiviral sensors in the cytoplasm. These sensors recognize viral nucleic acids and can initiate their destruction before infection of the target cell can be established. The capsiddisintegrating effect of these VHH antibodies can therefore be exploited for an antiviral therapy that protects cells against the virus.

[0051] The same applies to other anti-HIV-1 capsid antibodies disclosed herein, and in particular to Re39A08 and Re39E04, which target the capsid in a manner that keeps the capsid intact but prevents capsid entry into an FG phase, blocking passage through NPCs. Since this passage is crucial for an HIV-1 infection cycle and essential for the infection of non-dividing cells, these VHH antibodies can also protect cells against incoming virus, provided, the VHHs are expressed in the cells to be protected.

[0052] Thus, the here disclosed anti-VHH antibodies may be used for an anti-viral therapy that allows the immune system to eradicate the virus and thus to cure the infection. In certain embodiments, such treatment comprises somatic gene therapy, e.g., on hematopoietic stem cells, as detailed below.

[0053] Human cells expressing capsid-disintegrating or nuclear import-inhibitory anti-capsid antibodies, e.g., VHH antibodies should become refractory to HIV-1 infection. To achieve this, an appropriate anti-capsid antibody expression cassette can be integrated into the genome of the target cell by known methods such as lentiviral transduction, CRISPR / Cas9 gene editing or transfection with a linearized plasmid. Gene editing would have the advantage of directing the integration to a pre-defined locus. The expression cassette should be carefully designed for this purpose and, e.g., contain not only the antibody-coding region but also regulatory elements such as a promoter (of appropriate strength and cell type specificity) as well as a polyadenylation signal.

[0054] In certain embodiments, the approach comprises generating an HIV-1 resistant CD4+ T-cell population in an infected patient that cannot be depleted by virus infection. Once sufficiently expanded, this resistant T-cell pool would ensure a normal immune function and eventually allow the immune system to clear the infection.

[0055] CD4+ T-helper cells present a large variety of T-cell receptors (TCRs), which in turn are responsible for the proper recognition of antigens / pathogens in a process called antigen presentation (Murphy and Weaver, 2016). The TCR repertoire is not encoded genomically but is generated by a recombination process during T cell development. Therefore, the antibody expression cassette should be integrated into a precursor cell - before TCRs are diversified. Hematopoietic stem cells are therefore a suitable starting material for the integration of the antibody expression cassette. They may be derived from the patient itself or from an immunologically compatible donor.

[0056] The genomically integrated anti-capsid antibody can be regarded as a synthetic host restriction factor against HIV-1 . For a robust therapy against the rapidly mutating HIV- 1 virus, it is desirable to keep the probability of the virus escaping this restriction as low as possible. In this regard, targeting the CA protein is particularly advantageous, as it is the most conserved and genetically most constrained (fragile) of the 15 HIV-1 proteins (Rihn et al., 2013; Toccafondi et al., 2021 ). This fragility is likely due to the many different interactions CA has to functionally maintain within the viral capsid.

[0057] The probability of escaping restriction can be further reduced by “a second line of defense”. For example, a second VHH antibody can be incorporated into the same or a different expression cassette, targeting the capsid in a different way and thus making an eventually occurring escape mutation in the CA protein ineffective. In certain embodiments, the second antibody belongs to a different antibody class as shown in Table 1. In further embodiments, the second VHH may also bind another target such as the viral integrase, leading either to a neutralization, a mis-targeting, or a degradation of the enzyme.

[0058] In further embodiments, the anti-capsid antibody can be fused to an antiviral effector such as Trim5a. Human Trim5a (UniProt ID Q9C035) is a host restriction factor against several lentiviruses. It can form a cage around lentiviral capsids in the cytoplasm and target them for degradation. Trim5a, however, is ineffective against HIV-1 due to cyclophilin A blocking its recognition of the HIV-1 capsid (Ganser-Pornillos and Pornillos, 2019). A fusion with an anti-HIV capsid antibody should render Trim5a effective against an HIV-1 infection, not only by impairing NPC passage of the capsid- enclosed genome but also by effective competition with the much weaker cyclophilin A-binding, enabling Trim5a -mediated capsid degradation.

[0059] Antibodies

[0060] The present invention relates to an antibody targeting a lentiviral capsid assembly and antagonizing the partitioning of the capsid into an in vitro assembled FG phase.

[0061] The antibody may be any antibody comprising at least one polypeptide chain and having at least one antigen-binding domain.

[0062] In certain embodiments, the antibody is a single-chain antibody which consists of a single polypeptide chain. In certain embodiments, a single-chain antibody comprises a heavy chain or a fragment thereof fused on a light chain or a fragment thereof. In particular embodiments, a single-chain antibody comprises a heavy chain only. A single chain antibody comprises at least one antigen-binding domain, e.g., one or two antigen binding domains.

[0063] In certain embodiments, the antibody of the present invention is in a monovalent format, i.e. , it has a single binding site for a lentiviral capsid.

[0064] In more particular embodiments, the antibody is a single-domain antibody, i.e., an antibody which comprises a heavy chain only and binds to its target antigen with a single antigen-binding domain. In those embodiments, the antigen-binding domain comprises heavy chain variable domain CDRs only, particularly at least a heavy chain variable domain CDR3 and more particularly three heavy chain variable domains CDR1 , CDR2 and CDR3. A typical example of a single-domain antibody is a VHH antibody, e.g., a camelid-derived VHH antibody. In the following, the term “VHH antibody” encompasses all types of “single-domain antibodies” if not indicated otherwise. A single-domain antibody, e.g., a VHH antibody according to the present invention is characterized by (i) a CDR3 sequence, (ii) a combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, (iii) by a complete VHH sequence, or (iv) by competition with a specific reference antibody. Specific CDR and VHH sequences are provided in the Tables, Figures and the Sequence Listing.

[0065] A VHH antibody may be a monovalent heavy chain-only antibody comprising a CDR1 domain, a CDR2 domain and a CDR3 domain linked by framework regions including, but not being limited to, whole VHH antibodies, e.g., native VHH antibodies comprising framework regions derived from camelids, and modified VHH antibodies comprising modified framework regions, VHH antibody fragments and VHH antibody fusion proteins, e.g. a fusion protein with an immunoglobulin or non-immunoglobulin peptide or polypeptide, as long as it shows the properties according to the invention.

[0066] There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but there is no standard unequivocal method. Determination of CDR sequences from antibody heavy chain variable regions 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 embodiments of the present invention, the CDR sequences of a variable region are determined using the Kabat method. CDRs may also be defined through a multiple alignment (with many other VHH antibodies), to identify the hot-spots of variability and relate them to a standard VHH antibody structure. It is also possible to define CDRs by analyzing the structure of the VHH antibody and deciding what is a loop and what is the antibody’s scaffold. In some cases, CDR-adjacent residues are also variable, and are therefore included in the CDR definition.

[0067] The present invention is also directed to a covalent or non-covalent conjugate of an antibody molecule, e.g., a VHH antibody molecule to a to a heterologous moiety, which may be a non-proteinaceous structure or a heterologous polypeptide moiety. For example, the heterologous moiety may be a label, a capture group such a solid phasebinding group, or an effector.

[0068] In certain embodiments, the heterologous moiety is selected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide. In certain embodiments, the heterologous moiety is conjugated to the C-terminus of an antibody chain or a VHH antibody. These conjugates are e.g., suitable for diagnostic applications.

[0069] In particular embodiments, the heterologous moiety is an effector polypeptide. The effector may be a polypeptide which impedes the entry of the capsid into an FG phase as defined according to Frey et al. 2018, the content of which is herein incorporated by reference. In particular, it may be a negatively charged polypeptide moiety, e.g., an amino acid sequence having a length of 5-100 amino acids with a content of at least 20%, at least 35% or at least 50% negatively charged amino acids, i.e. , Glu and Asp, and optionally being depleted of FG-attractive residues, i.e. Trp, Tyr, Phe, Met, Cys, Leu, lie, Vai, His and Arg. In further embodiments, the effector polypeptide may be an antiviral effector polypeptide such as Trim5a, which is an E3 ubiquitin ligase that triggers degradation of the capsid by the proteasome or the autophagic pathway.

[0070] According to the present invention, sequences related to the above sequences are encompassed. These related sequences are defined by having a minimum identity to a specifically indicated amino acid sequence, e.g., a CDR or VHH sequence. This identity is indicated over the whole length of the respective reference sequence and may be determined by using well-known algorithms such as BLAST.

[0071] In particular embodiments, a related CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated CDR3 sequence, e.g., a substitution of 1 , 2, or 3 amino acids.

[0072] In particular embodiments, a related combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence has an identity of at least 80% or at least 90% or at least 95% to a specifically indicated combination of a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, e.g., a substitution of 1 , 2, 3, 4, 5 or 6 amino acids by different amino acids.

[0073] In particular embodiments, a related VHH sequence has an identity of least 70%, at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence, e.g., a substitution of 1 , 2, 3, 4, 5 or up to 20 amino acids.

[0074] Further, the invention refers to a VHH antibody, which competes with a specific VHH antibody disclosed herein for the binding to a lentiviral capsid. In certain embodiments, a competing VHH antibody binds the same or an overlapping epitope on the lentiviral capsid. For example, the invention refers to a VHH antibody, which competes with a reference antibody, e.g., VHH antibody Re39E04 having a VHH sequence as shown in SEQ. ID NO: 19, VHH antibody Re39B08 having a VHH sequence as shown in SEQ. ID NO: 31 , VHH antibody Re39G05 having a VHH sequence as shown in SEQ. ID NO: 35, VHH antibody Re39B09 having a VHH sequence as shown in SEQ. ID NO: 39, VHH antibody Re56H05 having a VHH sequence as shown in SEQ. ID NO: 43, VHH antibody Re56B09 having a VHH sequence as shown in SEQ. ID NO: 47, VHH antibody Re39G129 having a VHH sequence as shown in SEQ. ID NO: 51 , or VHH antibody KG11 D11 having a VHH sequence as shown in SEQ. ID NO: 55 for the binding to a HIV-1 capsid.

[0075] Competition may be determined by label-free biolayer interferometry performed as a cross-competition or epitope binning assay using a label-free detection system, e.g., an Octet® system from Sartorius, according to the manufacturer's instructions.

[0076] In particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1 , CDR2 or CDR3 sequence and / or an amino acid in a framework region, is replaced by another amino acid, while preserving structural integrity and epitope-binding of the VHH antibody. These exchanges can be conservative (i.e. , by a similar amino acid) or non-conservative.

[0077] In further particular embodiments, at least one amino acid of a reference sequence, including an amino acid in a CDR1 , CDR2 or CDR3 sequence and / or an amino acid in a framework region, is replaced by a conservative amino acid substitution, i.e. a substitution of an amino acid by another amino acid with similar biochemical properties, for example a substitution of an aliphatic amino acid, e.g. Gly, Ala, Vai, Leu, or lie, for another aliphatic amino acid; a substitution of a basic amino acid, e.g. His, Lys or Arg, against another basic amino acid or against Met; a substitution of an acidic amino acid or an amide thereof, e.g., Asp, Glu, Asn or Gin, against another acidic amino acid or an amide thereof; a substitution of an aromatic amino acid, e.g., Phe, Tyr or Trp, against another aromatic amino acid.

[0078] In certain embodiments, the VHH antibody is a humanized VHH variant which differs from the original VHH sequence by at least one framework mutation, e.g., by 1 to 15 or 1 to 10 framework mutations, wherein the sequence identity to a human immunoglobulin variable region, e.g., a human germline immunoglobulin variable region is increased. In particular embodiments, a humanized VHH variant comprises humanized framework regions wherein at least one amino acid, e.g.,1 to 10 amino acids in the framework regions, i.e., the regions outside the hypervariable CDR1 , CDR2 and CDR3 regions are replaced by other amino acids found in a human framework region.

[0079] Methods of humanizing antibody framework sequences are well known in the art as described in a review article by Rossotti et al. (FEBS J. 289 (2022), 4304-4327) and the citations listed therein, the contents of which are herein incorporated by reference. The humanization changes the framework but keeps the paratope intact. In particular embodiments, the combination of CDR1 , CDR2 and CDR3 sequences remains unaltered. Thus, a humanized VHH antibody variant of the present invention may comprise a combination of CDR1 , CDR2 and CDR3 sequences of the respective camelid VHH antibody and humanized framework regions.

[0080] In particular embodiments, a humanized VHH antibody variant of the present invention may comprise a VHH sequence as described herein wherein at least one and up to 15 or up to 10 amino acids of the original camelid framework sequence are replaced by amino acids present at a corresponding position in a framework sequence of a variable domain of a human antibody heavy chain, particularly of a human heavy IgG chain, e.g., a germline version of a variable domain of a heavy IgG chain such as HV323 (SEQ ID NO. 63). In even more particular embodiments, a humanized VHH antibody variant of the present invention comprises a VHH sequence selected from SEQ. ID NO: 15, 19, 23, 27, 31 , 35, 39, 43, 47, 51 , 55 or 59 wherein at least one, at least 2 or at least 3 of amino acids at position 1 , 11 , 14, 19, 23, 40, 63, 69, 72, 72, 76, 80, 84, 88, 107 and 110 has been replaced, if necessary, by an amino acid selected from E1 , L11 , P14, R19, A23, A40, S63, T69, R72, K76, Y80, N84, R87, A88, Q107 and L110 as shown in Fig. 11 (c.f. the line “Humanization”). Tolerability of individual amino acid replacements at individual positions may be determined in a straightforward manner by testing the characteristics of the VHH antibody variants as described in Example 5 (Affinity), Example 6 (Thermostability) and Example 7 (Inhibition of capsid entry) and Example 8 (Inhibition of capsid targeting) as herein described below.

[0081] In further particular embodiments, a humanized VHH antibody variant of the present invention comprises a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 16-18, 20-22, 24-26, 28-30, 32-34, 36-38, 40-42, 44-46, 48-50, 52-54, 56-58, or 60-62 and has an overall amino sequence identity of at least 80%, at least 90% or at least 95% and up to 99% to the VHH sequence of the respective VHH antibody as shown in Table 2, i.e., SEQ. ID NO: 15, 19, 23, 27, 31 , 35, 39, 43, 47, 51 , 55 or 59.

[0082] In further particular embodiments, the VHH antibody is selected from antibody Re39A08 having a VHH sequence as shown in SEQ. ID NO: 15 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 15 are replaced by another amino acid.

[0083] In further particular embodiments, the VHH antibody is selected from antibody Re39E04 having a VHH sequence as shown in SEQ. ID NO: 19 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 19 are replaced by another amino acid. In further particular embodiments, the VHH antibody is selected from antibody Re39H08 having a VHH sequence as shown in SEQ. ID NO: 23 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 23 are replaced by another amino acid.

[0084] In further particular embodiments, the VHH antibody is selected from antibody Re56F11 having a VHH sequence as shown in SEQ. ID NO: 27 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 27 are replaced by another amino acid.

[0085] In further particular embodiments, the VHH antibody is selected from antibody Re39B08 having a VHH sequence as shown in SEQ. ID NO: 31 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 31 are replaced by another amino acid.

[0086] In further particular embodiments, the VHH antibody is selected from antibody Re39G05 having a VHH sequence as shown in SEQ. ID NO: 35 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 35 are replaced by another amino acid.

[0087] In further particular embodiments, the VHH antibody is selected from antibody Re39B09 having a VHH sequence as shown in SEQ. ID NO: 39 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 39 are replaced by another amino acid.

[0088] In further particular embodiments, the VHH antibody is selected from antibody Re56H05 having a VHH sequence as shown in SEQ. ID NO: 43 or a VHH antibody, which is a variant thereof including a human VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 43 are replaced by another amino acid.

[0089] In further particular embodiments, the VHH antibody is selected from antibody Re56B09 having a VHH sequence as shown in SEQ. ID NO: 47 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 47 are replaced by another amino acid.

[0090] In further particular embodiments, the VHH antibody is selected from antibody Re39G12 having a VHH sequence as shown in SEQ. ID NO: 51 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 51 are replaced by another amino acid.

[0091] In further particular embodiments, the VHH antibody is selected from antibody KG11 D11 having a VHH sequence as shown in SEQ. ID NO: 55 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 55 are replaced by another amino acid.

[0092] In further particular embodiments, the VHH antibody is selected from antibody KG12D02 or a VHH antibody, which is a variant thereof including a humanized VHH variant. In certain embodiments, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ. ID NO: 59 are replaced by another amino acid.

[0093] Further, the present invention relates to a nucleic acid molecule, e.g., a DNA molecule, encoding an antibody, e.g., a VHH antibody as indicated above, or a vector, comprising said nucleic acid molecule as indicated above in operative linkage with an expression control sequence, particularly with a heterologous expression control sequence. Furthermore, the invention relates to a cell comprising a nucleic acid molecule or a vector as described above. In certain embodiments, the vector is a non-viral or viral vector for therapeutic use, e.g., a vector adapted for somatic gene therapy wherein a nucleic acid molecule encoding the antibody is integrated ex vivo or in vivo into the genome of a cell from a host organism, e.g., a mammalian subject and particularly a human subject. Integration into the genome may be carried out by well-known methods such as homologous recombination, transposon-mediated integration, CRISPR / Cas9 gene editing or lentiviral integration. In certain embodiments, the cell is a lymphocyte, particularly a CD4+ lymphocyte or a precursor cell thereof, more particularly wherein the precursor cell is a hematopoietic stem cell.

[0094] Binding to a lentiviral capsid

[0095] The antibody of the invention specifically targets a lentiviral capsid and inhibits partitioning of a lentiviral capsid species into an assembled FG phase as described in detail in the examples. In certain embodiments, the lentivirus is a human immunodeficiency virus (HIV) such as HIV-1 or HIV-2. In particular embodiments, the lentivirus is HIV-1 including variants thereof.

[0096] A typical lentivirus capsid consists of hexamers and pentamers of the capsid (CA) protein, e.g., the HIV-1 CA protein which is produced from HIV Gag protein by proteolytic cleavage. In certain embodiments, determination of antibody targeting of a lentivirus capsid is determined using a capsid-like particle (CLP).

[0097] The antibody binds to a lentivirus capsid in an assembled state. The inventors have identified VHH antibodies, which bind with high affinity to different epitopes on a HIV- 1 capsid as shown in Table 1 , supra.

[0098] In certain embodiments, the antibody has a binding affinity to a lentiviral CA protein- hexamer expressed as dissociation constant KD of about 5 nM or less, about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less when measured as described in the Examples in detail. In certain embodiments, binding of the antibody causes disintegration and / or destabilization of a lentiviral capsid, particularly a HIV-1 capsid, e.g., as shown in Figure 9 and the legend thereof in detail.

[0099] Inhibition of capsid-partitioning

[0100] In certain embodiments, the antibody reduces partitioning of a lentiviral capsid into an in vitro assembled FG phase to 5% residual FG partitioning or less, particularly to 2% residual FG partitioning or less, and more particularly to 1 % residual FG partitioning or less when measured inside an FG particle as described in the Examples in detail.

[0101] In certain embodiments, inhibition of a lentiviral capsid is quantitatively determined with a HIV-1 derived capsid and / or a capsid-like particle (CLP) as described in the Examples.

[0102] In certain embodiments, the antibody inhibits targeting of the lentiviral capsid species to a nuclear pore complex as indicated in Table 1 , supra.

[0103] Stability

[0104] For the intended therapeutic application, the antibodies, particularly the VHH antibodies should not only be highly potent in inhibiting capsid partitioning, but also, they should be developable as biological drugs. This includes that they are stable enough to survive a lengthy, large-scale production process as well as transportation and storage (ideally for years in liquid formulation) without aggregation or loss of activity.

[0105] This also includes that the VHH antibodies should be stable when produced in the cytoplasm of cells to be protected from a lentiviral infection. Stability implies here slow turnover and thus also a low level of antigen presentation.

[0106] A good predictor for stability is thermostability, which can be measured, e.g., by thermal shift assays or specifically by differential scanning fluorimetry. The present inventors have identified several thermostable or hyperthermostable VHH antibodies as shown in Table 1 , supra.

[0107] In a particular embodiment, the invention relates to an antibody, particularly a VHH antibody, which is stable, particularly thermostable, or hyperthermostable. Preferably, the antibody has a melting temperature of at least about 40°C, of at least about 50°C, of at least about 60°C, of at least 80°C or of at least about 95°C when measured under reducing conditions and / or a melting temperature of at least about 60°C, of at least about 80°C, of at least about 90°C, or of at least about 95°C when measured under non-reducing conditions. Melting temperatures are determined as described herein.

[0108] Sets of antibodies

[0109] In a further aspect, the present invention relates to a set of two or more different antibodies directed against a lentiviral antigen, particularly a HIV-1 antigen, wherein at least one of said antibodies targets a lentiviral capsid and inhibits partitioning of said capsid into an assembled FG phase. The set may comprise at least one antibody as specifically described herein, e.g., as shown in Table 1 , particularly at least one VHH antibody in a monovalent format.

[0110] The set may comprise at least 2, 3, 4 or more different antibodies, e.g., VHH antibodies. In such a set, the individual antibodies are present in suitable molar ratios. Typically, the molar ratios are in the range of about 2:1 to about 1 :2, particularly about 1.5:1 to about 1 :1.5, even more particularly about 1 :1. In certain embodiments, the antibody set may comprise a single composition wherein the antibodies in said set consist of a predetermined number of different species of antibodies. The antibody set may comprise a plurality of compositions each comprising a different antibody species. The set of the present invention may be free from other antibodies.

[0111] In certain therapeutic embodiments using sets of antibodies, a single copy of each nucleic acid molecule encoding an antibody may be integrated into the genome of a target cell. In certain embodiments, the set the set of antibodies comprises at least two, e.g., two, three or four different antibodies, each of them targeting a lentiviral capsid and inhibiting partitioning of said capsid into an assembled FG phase.

[0112] In particular embodiments, the set of antibodies comprises at least two, e.g., two, three or four complementary antibodies, i.e. , antibodies from different antibody classes as shown in Table 1 , supra. The complementary antibodies may be directed against different epitopes of the lentiviral capsid.

[0113] For example, the set of VHH antibodies comprises at least two, e.g., two, three or four VHH antibodies, selected from at least two, three or four different VHH antibody classes.

[0114] The set of antibodies may also comprise an antibody directed to a lentiviral antigen which is different from the capsid such as the viral integrase.

[0115] Sets of different VHH antibodies are useful for therapeutic and diagnostic applications as described herein in detail below.

[0116] Monovalent and multivalent antibodies

[0117] In certain embodiments, the antibody of the present invention is in a monovalent format, i.e., it has a single binding site for a lentiviral capsid. In these embodiments, the antibody may be present as such or covalently or non-covalently attached to a heterologous moiety, e.g., a peptidic or non-peptidic moiety.

[0118] In further embodiments, the antibody of the present invention is in a multimeric, e.g., dimeric, or trimeric format. In these embodiments, several VHH antibody units may be covalently or non-covalently attached to each other together via a linker and / or a multimerization, e.g., dimerization or trimerization moiety.

[0119] In certain embodiments, the antibody may be covalently or non-covalently conjugated to a heterologous moiety, which is selected from a labeling group, a capture group, or an effector group, and wherein the heterologous moiety is particularly selected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase, or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide. In further embodiments, the heterologous moiety is selected from human serum albumin, an albumin-binding moiety, or an Fc fragment of an immunoglobulin molecule, e.g., IgA, IgD, IgE, IgG, IgM, or a subtype thereof. In still further embodiments, the heterologous moiety is selected from one or several non-peptidic polymer moieties, preferably hydrophilic polymer moieties, such as polyethylene glycol (PEG).

[0120] In certain embodiments, the antibody is a homodimeric VHH antibody, wherein a VHH antibody unit is covalently attached to a dimerization moiety, e.g., an immunoglobulin IgG Fc fragment.

[0121] In certain embodiments, the antibody is a heterodimeric VHH antibody, particularly a covalently linked VHH heterodimer comprising a first VHH antibody and a second VHH antibody wherein the first VHH antibody and the second VHH antibody bind to different epitopes on HLA.

[0122] Therapeutic applications

[0123] Still a further aspect of the present invention is the use of an antibody, a nucleic acid molecule encoding said antibody, a vector comprising said nucleic acid molecule or a cell transformed or transfected with said vector in medicine, particularly for therapeutic use. In certain embodiments, the antibody, the nucleic acid molecule, the vector, or the cell is used in human medicine.

[0124] Therapeutic applications may be based on the direct production of the antibody in the cell to be protected from a virus infection within a subject in need thereof. In certain embodiments, nucleic acid molecule encoding the antibody or a vector comprising the nucleic acid molecule is introduced into a cell to be protected and integrated in its genome. Expression of the antibody within the cytoplasm provides immunity against an infection with a lentivirus, e.g., an infection with HIV-1. In certain embodiments, the nucleic acid molecule or the vector is introduced into a CD4+ lymphocyte or a precursor cell thereof, e.g., a hematopoietic stem cell.

[0125] In certain embodiments, the cell is obtained from the subject in need of being protected from a virus infection and re-introduced into the subject after being transformed or transfected with the nucleic acid molecule or the vector.

[0126] In therapeutic applications, the antibody, the nucleic acid molecule, the vector or the cell is provided in an effective amount to a subject in need thereof, particularly to a human subject. The dose will depend on the specific type of agent, the type of disease, and the route of administration.

[0127] Typically, the antibody, the nucleic acid molecule, the vector or the cell is administered as a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients are well- known in the art.

[0128] In certain embodiments, a single treatment provides a long-lasting cure.

[0129] Prophylactic use of nucleic acid molecules coding for the antibodies, is considered for a subject that is likely exposed to the pathogen and / or a subject that is immunocompromised.

[0130] The nucleic acid molecule, the vector, or the cell may be provided alone as a monotherapy or with a further active agent as a combination therapy, particularly with a further agent that is useful in the prevention, treatment, and / or mitigation of a disorder caused by and / or associated with an infection with a lentivirus such as HIV-1 . In certain embodiments, the nucleic acid molecule, the vector, or the cell is administered in combination with a further antiviral agent. Diagnostic applications

[0131] Diagnostic applications include in vitro methods wherein an antibody or a set of different antibodies is used for detecting a lentivirus such as HIV-1 in a sample, e.g., in a body fluid such as saliva, sputum, a lung lavage, a swab, blood, serum or plasma, stool samples or in a tissue or biopsy sample. In particular embodiments, the sample is blood, serum, or plasma.

[0132] Diagnostic applications further include in vivo methods wherein an antibody is used for detecting a lentivirus such as HIV-1 in a subject, particularly in a human patient. For diagnostic applications, the antibody may carry a label for direct detection or used in combination with secondary detection reagents, e.g., biotin / streptavidin, detection antibodies, including conventional antibodies or VHH antibodies, for indirect detection according to established techniques in the art.

[0133] The recombinant production of antibodies for diagnostic applications is well-known in the art. VHH antibodies including monomeric and multimeric VHH antibodies may be produced as described in WO 2022 / 023483 and WO 2022 / 023484, the contents of which are herein incorporated by reference, or by other methods well-known in the art.

[0134] Still a further aspect of the present invention is a method of recombinantly producing a VHH antibody by growing a suitable host cell in a culture medium and obtaining the VHH antibody from the cell or the culture medium. Suitable culture media and culture conditions are well known in the art.

[0135] Further, the present invention is explained in more detail by the following Figures and Examples. Figures

[0136] Figure 1 : Characterization of two types of HIV-1 capsids used in this application (A-C) Size exclusion chromatography (SEC) of 40 nm spheres and CLPs on a Superose 6 increase 10 / 300 GL column. Absorbance traces were recorded at 280 nm (to detect total protein) as well as at 490 nm (GFPs) or 580 nm (mCherry). SEC was the final step of the capsid preparations (as described in Examples 1 -2). Note that the 40 nm capsid spheres and CLPs (each with covalent EGFP tracer) eluted in the void volume. CLPs assembled in the presence of 2 mM non-fused mCherry also eluted in the void volume along with a fraction of the mCherry, indicating encapsulation.

[0137] (D) 40 nm capsid spheres (left) and capsid-like particles (CLPs, right) analyzed by electron microscopy after negative staining with 2% uranyl acetate on carbon-coated 300-mesh copper grids. Micrographs were collected on a FEI Tecnai G2 Spirit TWIN 120 kV transmission electron microscope equipped with a Gatan Ultrascan 2k x 2k CCD detector. Note that the analyzed capsids appear intact.

[0138] Figure 2: HIV-1 capsids behave like an NTR

[0139] (A) An FG phase was assembled as described previously (Ng et al., 2021 ) and in Example 3. It was tested for partitioning of mCherry, of unfused EGFP, an IBB-EGFP fusion, and the IBB-EGFP fusion with its IBB domain (importin [3-binding domain of importin a) being pre-bound to importin [3. Analysis was by confocal laser scanning microscopy. Note that mCherry remained firmly excluded from the FG phase. The presence of importin [3 shifted the IBB-EGFP fusion from exclusion (partition coefficient of 0.2) to strong intra-phase accumulation (partition coefficient of ~100).

[0140] (B) Experiment was performed as in panel A but 40 nm capsid spheres and CLPs, each labelled covalently with EGFP, were used as transport substrates. Both of them entered the FG phase to high partition coefficients of around 200-400. By contrast, free EGFP remained excluded (partition coefficient <0.1 ).

[0141] (C) Experiment was performed as in panel A, using the indicated transport substrates. sinGFP4a, a super-inert (i.e., highly FG-repellent) GFP variant (Frey et al., 2018) remained excluded from FG phases (partition coefficient <0.1 ), but covalently sinGFP4a-labelled 40nm spheres showed efficient partitioning into the FG phase (partition coefficient of ~500). As control, partially assembled capsomer (i.e., CA- hexamer / pentamer mixture) with sinGFP4a labeling only decorated the surface of the FG phase and failed to penetrate deeper into the phase.

[0142] (D) FG partitioning experiment with free mCherry or mCherry that had been encapsulated in CLPs during assembly. The CLP-encapsulated mCherry accumulated inside the FG to a partition coefficient >2 000 times higher than that of free mCherry (<0.1 ).

[0143] Figure 3: Sequences and CDR definition of anti-HIV 1 capsid VHH antibodies The Figure shows aligned sequences with CDRs being marked.

[0144] Figure 4: Anti-HIV-1 capsid VHHs block FG partitioning of 40 nm spheres

[0145] Experiment was performed as in Figure 2C, there difference being that the 40 nm capsid spheres (covalently labelled with sinGFP4a) had been pre-incubated with representative anti-capsid VHH antibodies as indicated. The VHH antibodies, in particular Re39A08 and Re39E04, drastically reduced the capsid partitioning, as indicated by the partition coefficients given below the images. For a more comprehensive overview of VHH effects, see Table 1 .

[0146] Figure 5: Anti-HIV-1 capsids VHH antibodies block FG partition of CLPs with a covalent GFP label

[0147] Experiment was performed as described in Figure 4 but partitioning of CLPs with a covalent GFP label was analyzed. Note the complete block of capsid entry by all anticapsid VHH antibodies tested. Re6B06 (Guttler et al., 2021 ) is an VHH directed against the receptor-binding domain of the SARS-CoV-2 spike protein and served here as a negative control.

[0148] Figure 6: FG phase entry of CLPs filled with mCherry is also blocked by anti- capsid VHHs

[0149] Experiment was identical to Figure 5 except that a different CLP assembly was used.

[0150] Figure 7: The FG phase entry block by anti-capsid VHH antibodies is enhanced by a fused negatively charged tail VHH Re39E04 was used either directly or after fusing a 25 residue peptide with a net charge of -11 to its C terminus. The fused peptide is FG-repellent. It diminished the residual interaction of the capsid with the FG phase strongly down to the limit of detection.

[0151] Figure 8: Anti-capsid VHHs block the targeting of capsid spheres to NPCs of permeabilized Hela cells

[0152] Details of the experiment are described in Example 8. Digitonin-permeabilized HeLa cells (with a perforated plasma membrane) were incubated with GFP-labelled fluorescent 40 nm capsid spheres and an Alexa647-labelled VHH directed against the NPC protein Nup133 for 30 minutes before imaging by confocal laser scanning microscopy. Note the co-localization of the capsid spheres with the NPC marker. The NPC signal of the GFP-labelled capsid spheres was greatly reduced in the presence of indicated anti-capsid VHHs (fused to an FG-repellant, negatively charged tail, as in Figure 7).

[0153] Figure 9: Identification of capsid-disintegrating VHH antibodies

[0154] CLPs were assembled as described above and in Example 2. They were then incubated with indicated VHH antibodies (VHH: CA monomer ratio = 1.5 : 1 ) at room temperature for 15 min, then all samples were applied to a glow-discharged carbon foil covered 400 mesh copper grid. After successive washing with water, samples were stained with 1 % uranyl acetate aq. and evaluated at room temperature using a Talos L120C transmission electron microscope (Thermo Fisher Scientific). Note that intact capsids are clearly detectable with VHHs Re39A08, Re39E04 or Re39H08, while Re39B09, Re39G05, Re39B12, and KG11 D11 converted the CLPs into smaller entities.

[0155] Figure 10: Capsid-disintegrating VHHs block FG phase entry of CLPs.

[0156] Assay was performed as described in Figure 5, using indicated VHHs as inhibitory agents.

[0157] Figure 11 : Sequence Alignment of VHH antibodies of the present invention with the human IgG germline sequence HV323_HUMAN. Preferred positions for humanization are indicated: E1 , L11 , P14, R19, A23, A40, S63, T69, R72, K76, Y80, N84, R87, A88, Q107 and L110 whereby the numbering corresponds to the human reference sequence.

[0158] Examples

[0159] Example 1 : Assembly of 40 nm capsid spheres with a covalent GFP tracer

[0160] The CA-P1A / N21 C / A22C mutant (Lau et al., 2020) (SEQ ID NO: 1 ) was expressed with an N-terminal His14-bdSUMO tag (SEQ ID NO: 2) (Frey and Gorlich, 2014) in Escherichia coH. Induction was with 0.5 mM IPTG (isopropyl-[3-thiogalactoside) at 18°C for 16 hours. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris / HCI pH 8.0, 500 mM NaCI, 20 mM imidazole, 1 mM TCEP (tris(2- carboxyethyl)phosphine), 2 mM PMSF (phenylmethylsulfonylfluoride), and lysed. The lysate was cleared by centrifugation at 8,500 x g for 30 min. The soluble fraction was incubated with Ni(ll) chelate beads for 1 hr at 4°C. The beads were subsequently washed with wash buffer (50 mM Tris / HCI pH 8.0, 40 mM imidazole, 300 mM NaCI, 1 mM TCEP). Proteins were eluted from the beads by tag cleavage with 100 nM of bdSENPI protease in cleavage buffer (50 mM Tris / HCI pH 8.0, 150 mM NaCI, 0.5 mM TCEP) for 3 hours at 4°C. CA [P1A / N21 C / A22C] fusions proteins with EGFP (SEQ ID NO: 3) or sinGFP4a (SEQ ID NO: 4) were produced the same way. Proteins were concentrated to approximately 15 mg / ml. Assembly was performed by dialyzing against 50 mM Tris / HCI pH 8.0, 1 M NaCI and 0.1 mM IP6 (inositol hexaphosphate) for 48 hours (Lau et al., 2020). 40 nm spheres were concentrated and isolated from unassembled CA by ultracentrifugation at 280 000 g for 2.5 hours. The pellet was washed and resuspended in assembly buffer. CA-spheres were further purified by size exclusion chromatography on a Superose6 Increase 10 / 300 GL column (Cytiva) equilibrated in 50 mM Tris / HCI pH 8.0, 500 mM NaCI and 0.5 mM IP6. Assembly was either performed with CA-EGFP or CA-sinGFP4a fusion added as a fluorescent tracer (at 1 / 5 molar ratio to unlabelled CA). Quality control was by negative stain electron microscopy as documented in Figure 1 D. Example 2: Assembly of capsid-like particles (CLPs) with a covalent GFP or a non-covalent mCherry tracer

[0161] CA [P1A mutant] (SEQ ID NO: 5) was expressed as a Hisu-bdSUMO fusion and purified as in Example 1 . The tag-free protein was concentrated to approximately 20- 30 mg / ml. Assembly into CLPs essentially followed a published protocol (Schirra et al., 2023). Briefly, the buffer of the CA protein was exchanged to 25 mM MES pH 6.0, 50 mM NaCI and 1 mM TECP, assembly was initiated by adding 0.5 volumes of 75 mM MES pH 6.0, 150 mM NaCI, 15 mM IP6, 3 mM TCEP, and allowed to proceed in a volume of 500 pl for two hours at 37°C and a CA concentration of 12 mg / ml. Assembled CLPs were pelleted in a microcentrifuge at 21 ,000 g for 10 minutes at 4 °C. The pellet was resuspended in 500 pl of 25 mM Tris / HCI pH 8.0, 150 mM NaCI, 1 mM IP6 and 1 mM TCEP, and centrifuged again at 21 ,000 g for 5 minutes. CLPs in the supernatant were further purified by size exclusion chromatography on a Superose6 Increase 10 / 300 GL column equilibrated in 25 mM Tris / HCI pH 8.0, 150 mM NaCI, 1 mM IP6 and 1 mM TCEP, where they eluted in the void volume. Assembly was performed either in the presence of 2 mM soluble mCherry (SEQ ID NO: 7) (as a non-covalently encapsulated tracer) or with CA-EGFP fusion (SEQ ID NO: 6) used as a tracer in a 1 :20 to 1 :5 molar ratio to the unlabelled CA.

[0162] Example 3: FG phase experiment with mCherry, IBB-GFP and importin

[0163] An FG phase was assembled from an FG repeat domain (SEQ ID NO: 8) comprising 52 repeats of the sequence GGLFGGNTQPAT (SEQ ID NO: 9) essentially as described previously (Ng et al., 2021 ). 2 pl of a 1 mM (—60 pg / pl) FG domain solution (in 2 M GuHCI) were rapidly diluted with 50 pl assay buffer (50 mM Tris / HCI pH7.5, 150 mM NaCI, 5 mM DTT). 7.5 pl of the resulting suspension were mixed with 22.5 pl substrate with 6 pM mCherry as an exclusion marker in assay buffer. Where indicated in Figure 2A, 3 pM EGFP, or 3 pM IBB-EGFP, or 1 pM of a pre-formed IBB- EGFP*lmportin 0 complex were also present. The resulting mixtures ([FG domain] =10 pM) were pipetted into collagen-coated 18-well p-slides (IBIDI, Germany). Prior to sequential confocal laser scanning at 561 nm and 488 nm, FG particles were allowed to sediment for 60 minutes to the bottom of the slide. Note that phase separation resulted in pm-sized, spherical FG particles, which excluded mCherry (Figure 2). The IBB-GFP fusion alone also remained excluded but accumulated to a partition coefficient of ~100 in the presence of importin [3. Quantitation was by integrating the fluorescent signal in the respective areas of confocal scan, using the Image J / Fiji software. Partition coefficients were calculated as the signal inside independent FG particles divided by the signal for reference areas in outside regions (in the buffer). For each image filed, raw signal within > 5 FG particles and 5 reference areas were quantified.

[0164] Example 4: Partitioning of HIV-1 capsid species into an FG phase

[0165] Experiments were performed as described in Example 3, the only difference being that different fluorescent transport substrates were added. As indicated in Figure 2B-D, these included: ( / ) 3 pM of non-fused free EGFP, sinGFP4a (super-inert, FG-phobic GFP variant; (Frey et al., 2018)), or mCherry; 0.4 pM of mixed pentameric / hexameric capsomer-sinGFP4a fusions; ( / / ) 10 nM of 40 nm spheres with covalent EGFP or sinGFP4a tracer (corresponding to 1.2 pM CA protein); (Hi) CLPs tagged with a covalent EGFP tracer (CA concentration = 1 .2 pM); (iv) CLPs non-covalently filled with mCherry (CA concentration = 1 .2 pM). The results are shown in Figure 2B-D. Note that all fully assembled capsid species (having diameters of 40 nm to >100 nm) partitioned very efficiently in the FG phase reaching partition coefficients of around 500. In contrast, the much smaller non-fused GFP or mCherry (diameter ~5 nm) remained firmly excluded. The partially assembled (hexameric) capsomer-sinGFP4a fusion only decorated the surface of the FG phase.

[0166] Example 5: Affinity of VHH antibodies for hexameric CA capsomers

[0167] VHH antibodies were expressed with an N-terminal DsbA signal peptide and with a C- terminal with a non-cleavable C-terminal Spacer-His tag of the sequence TSGSGTEGSEHHHHHHHHHHHH (SEQ ID NO: 10) in the periplasm of E. coli and released by an osmoshock, whereby pelleted bacteria where initially resuspended in 40% w / v sucrose, 75 mM Tris / HCI pH 8.0, 5 mM EDTA, shaken for one hour at room temperature, and then shocked by the addition of 10 volumes ice-cold water. After another 30 minutes incubation at 4°C, bacteria were pelleted again and the VHH were purified from the cleared supernatant by Ni(ll) chelate chromatography with imidazole elution.

[0168] For affinity measurements, biotinylated CA hexamers were produced using the CA P1A / A14C / E45C / W184A / M185A mutant (SEQ ID NO: 11 ). The CA protein was expressed as a His14-bdSUMO fusion and purified by Ni(ll) chelate chromatography with protease elution as described in Example 1 . A second version carried a C-terminal Avi-tag (SEQ ID NO: 12) that was biotinylated by incubation with E. coli BirA, biotin, and ATP (Beckett et al., 1999). For assembly, non-biotinylated and biotinylated CA mutant were mixed at a 5:1 molar ratio, and assembled in a high salt buffer (50 mM Tris / HCI pH 8.0, 1 M NaCI, 100 pM IP6) for 24 hours at a protein concentration of I Q- 15 mg / ml at room temperature. The sample was then dialyzed into 50 mM Tris / HCI pH 8.0. Hexamers were isolated by size exclusion chromatography (SEC) on a Superdex 200 Increase GL column (Cytiva) in 20 mM Tris / HCI pH 8.0, 150 mM NaCI, 100 pM IP6, and quality controlled by negative staining EM.

[0169] Bio-layer interferometry (BLI) experiments were performed using High Precision Streptavidin biosensors and an Octet RED96e instrument (ForteBio / Sartorius) at 25 °C with phosphate-buffered saline (PBS) pH 7.4, 0.02% (w / v) Tween 20 and 0.1 % (w / v) bovine serum albumin (BSA) as assay buffer. Biotinylated CA hexamers were immobilized on biosensors until a wavelength shift / binding signal of 1 nm was reached. The biosensors were dipped into wells containing 40, 20, 10, 5, 2.5 or 1.25 nM VHH for 10 minutes association, and then incubated with assay buffer for 30 minutes dissociation. Data were reference subtracted, and curves were fitted with a mass transport model (Octet Data Analysis HT 12.0 software). This provided estimates for the dissociation constants (KDs) listed in Table 1.

[0170] Example 6: Thermostability of VHH antibodies

[0171] To obtain the thermostability data listed in Table 1 , VHH antibodies were subjected to Differential Scanning Fluorimetry (DSF), which exploits the fact that thermal unfolding exposes aromatic / hydrophobic residues, which then bind and enhance the fluorescence of the added SYPRO Orange dye. Assays were performed in a volume of 20 l, at a VHH concentration of 1 mg / ml in 50 mM Tris / HCI, 150 mM NaCI (pH 8.0 at 20°C) and 1x dye (diluted from a 5000x stock; Life Technologies). Sample were pipetted into a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® ‘B’ Seal foil (Bio-Rad), briefly centrifuged to remove air bubbles, and placed into a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were first incubated at 20°C for 5 minutes. Then the temperature was increased to 95°C in 1 °C steps of 45 seconds each. At the end of each step, fluorescence was measured after excitation at 532 nm using a 555 nm long-pass filter. Melting temperatures were defined as the inflection point of the first melting peak. A VHH antibody was considered stable if its melting peak was lower than its initial fluorescence at 20°C. Measurements included VHH antibodies produced under reducing conditions with a reduced structural disulfide bond. This was meant to simulate their expression in the reducing cytosol of human cells.

[0172] Example 7: Inhibition of capsid entry into the FG phase by VHH antibodies

[0173] For these experiments, VHHs in a His-tag-free form were used. The VHHs shown in Figures 4-8 were expressed as His14-NEDD8 fusions in the cytoplasm of E. coli, purified by Ni(ll) chelate chromatography with elution by the tag-cleaving NEDP1 protease (Frey and Gorlich, 2014), removing the His14-NEDD8 tag. This was followed by post-production formation of the structural disulfide bond of the VHHs as recently described (Gorlich et al., 2023). Where indicated, they carried a C-terminal FG-phobic fusion to a negatively charged “acidic tail” of the sequence TSGSGTEGSEGGEGGEGGEGGEDEDED (SEQ ID NO: 13).

[0174] For inhibition, capsid species (indicated in the respective figures) corresponding to 1 .2 pM CA protein were pre-incubated as indicated with 1.5 pM VHH antibody, before subjecting them to the FG phase partitioning assays as described in Example 4. Figures 4-6 and Table 1 document that several of the VHHs drastically reduced the capsid partitioning, up to 500-fold in the case of Re39A08. A weak signal typically remained at the surface of the FG particles. This signal was further reduced by the fusion of the aforementioned acidic tail (Figure 7). Example 8: Inhibition of capsid-targeting to NPCs of digitonin-permeabilized HeLa cells by anti-capsid VHH antibodies

[0175] HeLa-K cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM, high glucose), supplemented with 10% fetal calf serum (FCS) and antibiotics (“AAS”, Sigma-Aldrich) on 8-well p-Slides (IBIDI, Germany) to 70% confluence. Plasma membranes were permeabilized by treating the cells with 30 pg / ml digitonin (water- soluble fraction) in transport buffer (20 mM HEPES / KOH pH 7.5, 110 mM potassium acetate, 5 mM magnesium acetate, 0.5 mM EGTA, 250 mM sucrose) for 3 mins at 25°C (with gentle shaking), followed by three washes in transport buffer (Ribbeck and Gorlich, 2001 ). Permeabilized cells were then incubated for 30 minutes with 40 nM Alexa647-labeled anti-Nup133 nanobodies and 40 nm capsid spheres (carrying covalent GFP label and corresponding to a 0.7 pM CA concentration). Analysis was by sequential confocal laser scanning microscopy, performed directly through the live samples and with sequential excitation. Where indicated, capsid spheres had been pre-incubated with 2 pM of indicated the VHH antibodies.

[0176] Sequences

[0177] SEQ ID NO:1

[0178] >CA-[P1A / N21C / A22C]

[0179] AIVQNIQGQMVHQAISPRTLCCWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGG HQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHN PPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNW MTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVL

[0180] SEQ ID NO: 2

[0181] >H14-bdSUMO-CA-[P1A / N21C / A22C]

[0182] MSKHHHHSGHHHTGHHHHSGSHHHSGSAAGGEEDKKPAGGEGGGAHINLKVKGQDGNE VFFRIKRSTQLKKLMNAYCDRQSVDMTAIAFLFDGRRLRAEQTPDELEMEDGDEIDAMLHQT GGAIVQNIQGQMVHQAISPRTLCCWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTV GGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMT H N PPI PVGEI YKRWI I LGLN KI VRMYSPTSI LDI RQGPKEPFRDYVDRFYKTLRAEQASQEVKN WMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVL

[0183] SEQ ID NO:3

[0184] >CA-[P1A / N21C / A22C]-EGFP fusion AIVQNIQGQMVHQAISPRTLCCWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH QAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNP PI PVGEI YKRWI I LGLN KI VRMYSPTSI LDI RQGPKEPFRDYVDRFYKTLRAEQASQEVKN WM TETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLMVSKGEELFTGVVPIL VELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDH MKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGH KLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYL

[0185] STQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0186] SEQ ID NO: 4

[0187] >CA-[P1A / N21C / A22C]-Sin4aGFP fusion

[0188] AIVQNIQGQMVHQAISPRTLCCWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGG

[0189] HQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHN

[0190] PPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNW

[0191] MTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLTSKGEELFTGTVPI

[0192] KVELDGDVNGHKFSVKGEGEGDATEGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFAKYP

[0193] DHMKKHDFFKSAMPEGYTQERTIEFKDDGTYKTKAEVKFEGDTLVNKIELKGDDFKEDGNIL

[0194] GHKLEYNHNSHDVKIEADKEKNGIKANFKIKHNVEDGSEQEADHKQENTPIGDGPVKLPDN

[0195] HTLSTQTTLSKDPNEKKDHMVLKETVTAAGITKGEDEKDK

[0196] SEQ ID NO: 5

[0197] >CA-[P1A]

[0198] AIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH QAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNP PI PVGEI YKRWI I LGLN KI VRMYSPTSI LDI RQGPKEPFRDYVDRFYKTLRAEQASQEVKN WM TETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVL

[0199] SEQ ID NO: 6

[0200] >CA-[P1A]-GFP

[0201] AIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH QAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNP PI PVGEI YKRWI I LGLN KI VRMYSPTSI LDI RQGPKEPFRDYVDRFYKTLRAEQASQEVKN WM TETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLMVSKGEELFTGVVPI LVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYP DHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL GHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0202] SEQ ID NO: 7

[0203] >mCherry

[0204] SKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAW

[0205] DILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI

[0206] YKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKT

[0207] TYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0208] SEQ ID NO: 8 >GLFG 52x12

[0209] MQHHSHHGHHSHHGHHGHHGHHGHHGHHGSGGLFGGNTQPATGGLFGGNTQPATGGLF

[0210] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0211] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0212] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0213] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0214] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0215] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0216] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0217] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0218] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLF

[0219] GGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATGGLFGGNTQPATSC

[0220] SEQ ID NO: 9

[0221] GGLFGGNTQPAT

[0222] SEQ ID NO: 10

[0223] TSGSGTEGSEHHHHHHHHHHHH

[0224] SEQ ID NO: 11

[0225] >CA P1A, A14C, E45C, W184A, M185A mutant (for CA hexamers)

[0226] AIVQNIQGQMVHQCISPRTLNAWVKVVEEKAFSPEVIPMFSALSCGATPQDLNTMLNTVGGH QAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNP PI PVGEI YKRWI I LGLN KI VRMYSPTSI LDI RQGPKEPFRDYVDRFYKTLRAEQASQEVKNAAT ETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVL

[0227] SEQ ID NO: 12

[0228] >CA P1A,A14C,E45C,W184A,M185A mutant with C-terminal Avi-tag

[0229] AIVQNLQGQMVHQCISPRTLNAWVKWEEKAFSPEVIPMFSALSCGATPQDLNTMLNTVGG

[0230] HQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHN

[0231] PPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAA

[0232] TETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLSSGSGLNDIFEAQKIE

[0233] WHE

[0234] SEQ ID NO: 13

[0235] TSGSGTEGSEGGEGGEGGEGGEDEDED

[0236] SEQ ID NO: 14

[0237] >Re6B06

[0238] QVQLVESGGGLVQAGGSLRLSCAASGRAFSSAPMSWFRQAPGKEREFVASVSWSGDSTN

[0239] YADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCKRGPYWGQGTQVTVSS

[0240] New VHH antibodies:

[0241] SEQ ID NO: 15 >Re39A08

[0242] QVQLVESGGGLVQAGGSLTLSCTASGRTLSEYNMGWFRQAPGKEREFGAAITWTGLTRNY ADSVKGRFTVSRDNAKNTMYLQMNSLKPEDTAVYYCAARTAPLAETAYDRQNYYDQWGQG TQVTVSS

[0243] SEQ ID NO: 19

[0244] >Re39E04

[0245] QVQLVESGGGLVQAGGSLTLSCAASGRTLSEYNMGWFRQAPGKEREFVAAISWSSLTRNY ADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCAARTAPLPETAYGRQNYYDQWGQG TQVTVSS

[0246] SEQ ID NO: 23

[0247] >Re39H08

[0248] QVQLVESGGGLVQAGGSLTLSCAASGRTLSEYDMGWFRQAPGKEREFGAYISWTGLTRFY ADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAARTAPLAETAYDRQNYYDQWGQG TRVTVSS

[0249] SEQ ID NO: 27

[0250] >Re56F11

[0251] QVQLVESGGGLVQAGGSLTLSCTASGRTLSEYNMGWFRQAPGKEREFGAAITWTGLTKNY ADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAARTAPLTETAYDRQNYYDQWGQG TQVTVSS

[0252] SEQ ID NO: 31

[0253] >Re39B08

[0254] QVQLVESGGGSVQTGGSLRLSCAASGSAFMPYVMGWYRQSPGKQRELVAVISSGGPANYA

[0255] DSVKGRFTISGDNARNTVHLQMNSLKPEDTAVYYCNAKTYSRPYDYWGQGTQVTVSS

[0256] SEQ ID NO: 35

[0257] >Re39G05

[0258] QVQLVESGGGLVQPGGSLRLSCAASGFTFSKYAMSWYRQAPGKERELVASITSTGGATNYA DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVRDDLLLPHADRDFGSWGQGTQVT VSS

[0259] SEQ ID NO: 39

[0260] >Re39B09

[0261] QVQLVESGGGLVQPGGSLRLSCAASGFTFSKWKMSWYRQAPGKERELVAAITSTGVTTKY ADSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCNARDDLYSDYFEVGSWGQGTQVTV SS

[0262] SEQ ID NO: 43

[0263] >Re56H05

[0264] QVQLVESGGGLVQAGGSLTLSCAASGSTLSNPPMAWSRQAPGKEREWVAFISSGGITKYAD

[0265] PVKGRFTISRANAKNTVYLQMNSLKPEDTAVYYCNTEDYFGQGTQVTVSS

[0266] SEQ ID NO: 47

[0267] >Re56B09

[0268] QVQLVESGGGLVQAGGSLRLSCAASRSTFRFYFMGWYRQAPGKQRDFVADIRSDGTTNYA

[0269] DSVKGRFTISSDNGKNTVALQMDSLKPEDTAVYYCQARSLGFGEFDTYWGQGTQVTVSS

[0270] SEQ ID NO: 51 >Re39G12 QVQLVESGGGLVQPGGSLRLSCAASGSIFSINIMGWYRQAPGKQRELVATSAGGDRTNYAD

[0271] SVKGRFTISRDNAKNTVYLQMNSLNSEDTAVYYCFAEGFTYSRGGSYYSTPTWGAGTQVTV

[0272] SS

[0273] SEQ ID NO: 55

[0274] >KG11D11

[0275] QVQLVESGGGLVQAGGSLRLSCAASGLTSSAFTLGWRRQAPGKESELVGYIISGGSPQYAD

[0276] SVKGRFTISRDYAKNTLTLQMNSLKPEDTAVYYCYAYLSSADHYLGQGTLVTVSS

[0277] SEQ ID NO: 59

[0278] >KG12D02

[0279] QVQLVESGGGSVQAGGSLRLSCAASGLTSSAFTLGWLRQAPGKERELVGYIMSGGSPQYA

[0280] DSVKGRFSISRDYAANTLTLQMNSLKPEDTAVYYCYAYLSSADHYWGQGTQVTVSS

[0281] SEQ ID NO: 63

[0282] >HV323

[0283] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY

[0284] ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAEYFQHWGQGTLVTVSS

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Claims

Claims1. An antibody specifically targeting a lentiviral capsid wherein said antibody inhibits partitioning of said capsid into an assembled FG phase.

2. The antibody of claim 1 which reduces partitioning of a lentiviral capsid species into an in vitro assembled FG phase to 5% residual FG partitioning or less, particularly to 2% residual FG partitioning or less, and more particularly to 1% residual FG partitioning or less when measured inside an FG particle.

3. The antibody of claim 1 or 2, wherein the binding affinity to a lentiviral CA protein-hexamer expressed as dissociation constant KD is about 5 nM or less, about 1 nM or less, about 0.5 nM or less, or about 0.1 nM or less.

4. The antibody of any one of the preceding claims wherein said antibody inhibits targeting of the lentiviral capsid to a nuclear pore complex.

5. The antibody of any one of the preceding claims which causes destabilization and / or disintegration of a lentiviral capsid.

6. The antibody of any one of the preceding claims which is a single domain antibody, particularly a VHH antibody.

7. The antibody of any one of the preceding claims wherein the lentivirus is an HIV-1 strain.

8. The antibody of any one of the preceding claims, comprising(a) a combination of CDR1 , CDR2 and CDR3 sequences as shown in SEQ. ID NO: 16-18, 20-22, 24-26, 28-30, 32-34, 36-38, 40-42, 44-46, 48-50, 52-54, 56- 58, or 60-62 or(b) a combination of CDR1 , CDR2 and CDR3 sequences which has an identity of at least 80%, at least 90% or at least 95% to a combination of CDR1 , CDR2 and CDR3 sequences of (a).

9. The antibody of any one of the preceding claims, comprising(a) a VHH sequence as shown in SEQ. ID NO: 15, 19, 23, 27, 31 , 35, 39, 43,47, 51 , 55 or 59, or(b) a VHH sequence, which has an identity of at least 80%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a), particularly a humanized variant of the VHH sequence of (a).

10. The antibody of any one of the preceding claims which is stable, particularly thermostable, or hyperthermostable and(i) which has a melting temperature of at least about 40°C, of at least about 50°C, of at least about 60°C, of at least 80°C or of at least about 95°C when measured under reducing conditions, and / or(ii) which has a melting temperature of at least about 60°C, of at least about 80°C, of at least about 90°C, or of at least about 95°C when measured under non-reducing conditions.

11. A set of two or more different antibodies directed against a lentivirus antigen, particularly a HIV-1 antigen, wherein at least one of said antibodies targets a lentiviral capsid and inhibits partitioning of said capsid into an assembled FG phase.

12. A nucleic acid molecule encoding an antibody according to any one of claims 1 - 10, preferably in operative linkage with a heterologous expression control sequence, or a vector comprising said nucleic acid molecule.

13. A recombinant cell or non-human organism transformed or transfected with a nucleic acid molecule or a vector according to claim 12.

14. The recombinant cell of claim 13 which is a lymphocyte, particularly a CD4+ lymphocyte or a precursor cell thereof, more particularly wherein the precursor cell is a hematopoietic stem cell.

15. The antibody of any one of claims 1 -10, the set of claim 11 , the nucleic acid molecule or the vector of claim 12 or the cell of claim 13 or 14 for use in therapy, particularly wherein the antibody is directedly produced in a cell to be protected.

16. The antibody of any one of claims 1 -10, the set of claim 11 , the nucleic acid molecule or the vector of claim 12 or the cell of claim 13 or 14 for use in the prevention, treatment and / or mitigation of a condition caused by, associated with and / or accompanied by an infection with a lentivirus, particularly wherein the lentivirus is HIV-1 .

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