Nanobodies for cancer therapy

Abstract

The present disclosure provides single domain antibodies (VHHs or nanobodies) and conjugates thereof that can inhibit the binding of PD-L1 to PD-1 with high efficiency. The present disclosure further relates to viral vectors comprising the nanobodies or nanobody conjugates, and the use of the nanobodies, nanobody conjugates, and vectors for use in the treatment of cancer.

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application No. 22383011.8, filed October 20, 2022.

[0002] The present disclosure relates to the field of cancer therapy, and in particular to the development of new immune checkpoint inhibitors for the treatment of cancer. [Background technology]

[0003] Tumor cells use many strategies to evade the immune system, including engaging immune checkpoint (IC) pathways that induce immunosuppressive functions. Among various IC receptors, T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) have been used as therapeutic targets in various cancers. Several monoclonal antibodies (mAbs) that block these IC pathways (termed IC inhibitors or ICIs) have been shown to induce potent antitumor effects in a small subset of patients, leading to significant and durable clinical responses. Antibodies capable of blocking the PD-1 / PD-L1 axis, such as nivolumab, pembrolizumab, and atezolizumab, have shown remarkable therapeutic efficacy in patients with various tumor types [Xiang Z, et al., Front Pharmacol. 2022]. However, frequent adverse effects observed in patients treated with checkpoint inhibitors and a lack of response in some tumor types necessitate improvements in these treatments [Sun G, et al., Int J Oncol. 2022].

[0004] The sustained clinical benefit of these drugs is mainly limited by primary resistance to these treatments, or acquired resistance after initial response.In addition, because the receptor constitutes a natural mechanism for maintaining self-tolerance, systemic administration of ICIs leads to immune-based attacks on normal tissues in a significant proportion of patients.These immune-related adverse events (irAEs) can potentially affect any organ in the body, with dermatitis and thyroiditis being the most common, followed by other major concerns such as pneumonitis, hepatitis-colitis, hepatitis, hypophysitis, nephritis, myositis and adrenal inflammation.

[0005] Thus, there is a clear need to generate safer and more effective IC inhibitors, ideally for the treatment of cancer in an even higher proportion of patients. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Xiang Z,et al,Front Pharmacol.2022 [Non-patent document 2] Sun G, et al, Int J Oncol.2022 Summary of the Invention

[0007] The present inventors have developed novel IC inhibitors that overcome many of the drawbacks mentioned above. In particular, the present inventors have developed single domain antibodies (also referred to herein as VHHs or nanobodies) that can inhibit the interaction between programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) for their human and mouse orthologs.

[0008] Accordingly, a first aspect of the present disclosure relates to a single domain antibody comprising the following complementarity determining regions (CDRs): (i) a CDR1 described by SEQ ID NO: 1, a CDR2 described by SEQ ID NO: 2, and a CDR3 described by SEQ ID NO: 3, or (ii) a CDR1 described by SEQ ID NO: 5, a CDR2 described by SEQ ID NO: 6, and a CDR3 described by SEQ ID NO: 7.

[0009] As shown in the examples below, nanobodies comprising the CDRs of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 bind to PD-L1 and are therefore anti-PD-L1 nanobodies, whereas nanobodies comprising the CDRs of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 bind to PD-1 and are therefore anti-PD-1 nanobodies. These novel anti-PD-1 and anti-PD-L1 nanobodies have been shown to block the binding of human PD-L1 to PD-1. The inhibitory activity of the nanobodies described herein is surprising because the nanobodies contain only three (not six) CDRs for antigen binding. The examples below further demonstrate that this inhibitory activity translates into anti-tumor effects in MC38 colon adenocarcinoma tumors.

[0010] Furthermore, the disclosed nanobodies have several advantages over known anti-PD-L1 and anti-PD-1 antibodies. For example, the nanobodies of the first embodiment exhibit cross-reactivity with mouse and human PD-L1 and PD-1, inhibiting the binding of PD-1 to both species of PD-L1. Cross-reactivity is a desirable feature for translation studies, allowing animal studies to more smoothly translate to clinical settings. An additional advantage is that, due to their relatively small size, these nanobodies enter tumors and are more efficiently distributed within them. They also have manufacturing advantages, as they are easier and more cost-effective to produce. Nanobodies have been successfully expressed in a variety of systems, including bacteria, yeast, plant cells, insect cells, and mammalian cells, with high yields. This is possible due to their high solubility and physicochemical stability, their single-domain nature, and the fact that no post-translational modifications are required. The possibility of using inexpensive systems, such as E. coli, is a major advantage for cost-effective industrial scale-up. Nanobodies are also easier to handle for preparing pharmaceutical or diagnostic formulations.

[0011] A second aspect of the present disclosure relates to a nanobody conjugate comprising a nanobody as defined in the first aspect.

[0012] The inventors have found that, while monomeric nanobodies have already demonstrated anti-tumor efficacy comparable to known antibodies, fusing nanobodies to another molecule to form nanobody conjugates offers several advantages. For example, conjugating the nanobodies defined above to an immunoglobulin G (IgG) fragment crystallizable (Fc) domain surprisingly improved the PD-1 / PD-L1 inhibitory profile. This is demonstrated in the examples of the present disclosure, which show that conjugating two copies of the same nanobody to an immunoglobulin G fragment crystallizable (Fc) domain surprisingly improved the PD-1 / PD-L1 inhibitory profile and promoted even more potent anti-tumor activity in a mouse model of colorectal cancer compared to conventional antibodies. In this sense, the following examples show that homodimerization of the nanobodies of the first embodiment with an IgG Fc domain resulted in approximately 8-fold and 40-fold lower IC50 values ​​in PD-1 / PD-L1 binding assays for anti-PD-L1 nanobodies and anti-PD-L1 nanobodies, respectively.

[0013] In another example, it was further found that nanobody conjugates comprising IL-12 (a cytokine with potent anti-tumor activity) and one of the nanobodies described herein exhibit strong synergistic effects. Without wishing to be bound by theory, the inventors believe that this may be because IL-12 induces the production of IFN-gamma, which in turn induces the expression of PD-L1 and may limit the activity of the cytokine. The advantages of these nanobody-cytokine nanobody conjugates (also referred to herein as immunocytokines or ICKs) may be (i) a synergistic effect on the actions of IL-12 and anti-PD-1 nanobodies or anti-PD-L1 nanobodies, (ii) enrichment of IL-12 activity in the tumor due to high levels of PD-L1 and PD-1 in the tumor, which promote ICK retention in tumor tissue by interacting with the nanobody moiety, and (iii) reduced potential toxicity of IL-12 that becomes retained in tumor tissue.

[0014] We have also found that the nanobodies and nanobody conjugates defined above can be successfully delivered using replication-deficient alphavirus vectors based on Semliki Forest virus (SFV). As shown in the examples below, delivery of the nanobodies described herein via SFV vectors not only enabled high local intratumoral expression of the nanobody or nanobody conjugate, but also elicited important co-adjuvant responses that synergized with the immunostimulatory and antitumor activity of the nanobody. Delivery of nanobodies via viral vectors offers additional advantages in terms of manufacturing processes and regulatory requirements for clinical translation. Furthermore, a notable feature is that a single, extremely short-term administration of a vector locally expressing a dimerized nanobody was able to promote a potent and long-lasting antitumor response.

[0015] SFV-based RNA viral vectors further exhibit several advantages over other vectors for cancer therapy, such as higher expression levels, broad tropism, induction of immunogenic apoptosis in tumor cells, and the ability to elicit a strong IFN-I response.

[0016] Thus, a third aspect of the present disclosure relates to a polynucleotide encoding a Nanobody as defined in the first aspect, or a Nanobody conjugate as defined in the second aspect, and a fourth aspect relates to an expression vector comprising a polynucleotide as defined in the third aspect.

[0017] A fifth aspect of the present disclosure relates to a host cell comprising a polynucleotide as defined in the third aspect, or an expression vector as defined in the fourth aspect.

[0018] A sixth aspect relates to a viral particle comprising a polynucleotide as defined in the third aspect, or an expression vector as defined in the fourth aspect.

[0019] A seventh aspect relates to a method of producing a Nanobody or Nanobody conjugate as defined in the first and second aspects, or a method of producing a viral particle as defined in the sixth aspect, comprising: (a) culturing a host cell according to the fifth aspect under conditions suitable for producing the Nanobody, Nanobody conjugate or viral particle, thereby obtaining a culture containing the Nanobody, Nanobody conjugate or viral particle; (b) isolating or recovering the nanobody, nanobody conjugate or viral particle from the culture; The present invention provides a method comprising:

[0020] An eighth aspect relates to a pharmaceutical composition comprising a Nanobody, or a Nanobody conjugate, or an expression vector, or a host cell, or a viral particle, all as defined in the above aspects, together with a pharmaceutically acceptable excipient and / or carrier.

[0021] The present disclosure also provides, in a ninth aspect, a pharmaceutical composition, or a nanobody, or a nanobody conjugate, or an expression vector, or a host cell, or a viral particle, any of which is defined in the above aspects, for use in therapy. This aspect can be translated as use of a pharmaceutical composition, or a nanobody, or a nanobody conjugate, or an expression vector, or a host cell, or a viral particle, any of which is defined in the above aspects, for the preparation of a medicament. Also disclosed is a method of treatment comprising administering to a subject in need thereof a pharmaceutical composition, or a nanobody, or a nanobody conjugate, or an expression vector, or a host cell, or a viral particle, any of which is defined in the above aspects.

[0022] A tenth aspect provides a pharmaceutical composition, or a nanobody, or a nanobody conjugate, or an expression vector, or a host cell, or a viral particle, any of which is defined in the above aspects, for use in treating cancer. This aspect can be translated as use of a pharmaceutical composition, or a nanobody, or a nanobody conjugate, or an expression vector, or a host cell, or a viral particle, any of which is defined in the above aspects, for the preparation of a medicament for treating cancer. Also disclosed is a method of treating cancer comprising administering to a subject in need thereof a pharmaceutical composition, or a nanobody, or a nanobody conjugate, or an expression vector, or a host cell, or a viral particle, any of which is defined in the above aspects.

[0023] An eleventh aspect of the present disclosure provides the use of a nanobody or nanobody conjugate according to the first and second aspects for: (i) to detect human PD-L1 molecules and / or human PD-1 molecules; (ii) for flow cytometry assays; (iii) for cellular immunofluorescence detection; or (v) To diagnose cancer.

[0024] A twelfth aspect of the disclosure provides a method for detecting PD-L1 protein and / or PD-1 protein in a sample, the method comprising: (1) contacting a sample with a nanobody or nanobody conjugate according to the first and second aspects; (2) detecting antigen-antibody complexes, wherein the detected complexes indicate the presence of PD-L1 protein or PD-1 protein. Includes:

[0025] Finally, a thirteenth aspect relates to a diagnostic agent comprising a nanobody or nanobody conjugate according to the first and second aspects. [Brief explanation of the drawings]

[0026] [Figure 1]Figure 1. Expression of anti-PD-1 and anti-PD-L1 nanobodies from SFV vectors in vitro and anti-tumor activity in vivo. (A) Diagram of SFV vector encoding a monomeric nanobody (Nb), with the protein product shown on the right (not to scale). The subgenomic promoter (sgPr) enabling transcription of the subgenomic RNA encoding the Nb is indicated. (B-C) BHK-21 cells were infected with SFV virions expressing the indicated transgenes at an MOI of 20 or mock-infected and analyzed by Western blot at 24 hours using an anti-HA antibody (B) and specific PD-1 or PD-L1 binding ELISA for quantification (percentages of each fraction are indicated above the bars) (C). (D and E) The antitumor activity of SFV vectors encoding the monomeric anti-PD-1 (SFV-Nb11) nanobody (D) and the monomeric anti-PD-L1 (SFV-Nb6p) nanobody (E) was evaluated in an MC38 subcutaneous tumor model, using SFV-LacZ and saline as controls. Mice were intratumorally administered a single dose of 3 × 10 viral particles. Left graph: tumor growth after treatment. Data represent the mean ± SEM (n = 7 / group) and show a representative experiment of two with similar results. Right graph: survival curves of treated animals. UTR, untranslated region; HA, hemagglutinin tag; S, supernatant; CE, cell extract; ns, not significant. Magnification (B), 400x. [Figure 2]Figure 2. In vitro expression of nanobody-Fc fusion proteins from SFV vectors compared with conventional antibodies. (A) Schematic diagram of SFV vectors encoding nanobodies (Nb) against PD-1 and PD-L1 (SFV-Nb11-Fc and SFV-Nb6p-Fc, respectively) and conventional full-length antibodies (mAbs) against mouse PD-1 and mouse PD-L1 (SFV-aPD1 and SFV-aPDL1, respectively) fused to the indicated mouse IgG (mIgG) Fc domain. On the right, a schematic diagram of the various antibodies and their estimated molecular weights are shown. (B-C) BHK-21 cells were infected with SFV virions expressing the indicated transgenes at an MOI of 20 or mock-infected and analyzed at 24 h by specific IgG ELISA (percentages of each fraction are indicated above the bars) (B) and Western blot using antibodies against mouse IgG, SFV replicase, and α-actin (C). UTR, untranslated region; DTT, dithiothreitol; HC, IgG heavy chain. Magnification (B), 400x. [Figure 3] Figure 3. Inhibition of PD-1 / PD-L1 binding in vitro. Inhibition curves were performed using purified nanobodies against PD-1 (A) and PD-L1 (B). Monomeric nanobodies, nanobodies fused to the Fc domain, or commercially available antibodies were included in each assay. Mouse (left) or human (right) PD-1 / PD-L1 ectodomains were used. Data represent the mean ± SD of the percentage of PD-1 / PD-L1 binding, considering wells without blocking antibody as 100% binding. [Figure 4]Figure 4. Antitumor activity of SFV vectors encoding nanobody-Fc fusion proteins. The antitumor activity of SFV vectors expressing nanobodies or mAbs against PD-1 (A) and PD-L1 (B) was tested in the MC38 subcutaneous tumor model. When tumors reached a size of approximately 20 mm, a single intratumoral dose of 3 × 10 viral particles of the indicated vector was administered. SFV-LacZ and saline were used as controls. A and B: Left graph, tumor growth progression (mean ± SEM, one representative experiment out of two with similar results shown, n = 6–8 / group); middle graph, survival rate (pooled data from two independent experiments, n = 13–15 / group); right graph, tumor growth progression in cured mice rechallenged 2–3 months later with 5 × 10 MC38 cells in the left flank. Naive, untreated mice were included as controls. *, p<0.05; **, p<0.01; ****, p<0.0001; ns, not significant. [Figure 5] Figure 5. In vivo nanobody expression from intratumorally administered SFV vectors. Mice bearing subcutaneous MC38 tumors measuring approximately 40 mm were intratumorally injected with 3 x 10 SFV viral particles encoding monomeric nanobodies or Fc-fusion nanobodies (n = 4–5 per group). Mice were sacrificed 1 or 5 days later to assess in vivo nanobody expression. (A) Nanobody levels in tumors normalized by total protein content. (B) Nanobody levels in serum. (C) Levels of monomeric Nb11 nanobody and monomeric Nb6p nanobody in urine samples. (D) Urinary Nb11-Fc levels. *, p<0.05; ****, p<0.0001; ns, not significant. [Figure 6]Figure 6. Characterization of immune cell infiltration in MC38 tumors treated with an SFV vector encoding Nb6p-Fc. Mice (n = 5–6 per group) bearing approximately 50 mm3 MC38 tumors were administered a single intratumoral dose of 3 × 108 SFV viral particles. Five days later, they were sacrificed and immune cell infiltration was analyzed by flow cytometry. (A) Tumor growth curves after treatment and tumor weights at the time of sacrifice (day 5 after treatment). (B–D) Analysis of CD8+ T cell populations in tumor samples. (B) CD8+ T cell infiltration and expression of activation markers. (C) Analysis of MuLV-specific CD8+ T cells. (D) PD-1 expression on CD8+ T cells and MuLV-specific CD8+ T cells. (E) Analysis of various innate immune cell populations in tumor samples. Asterisks above the bars indicate comparison with the saline group. One sample from SFV-LacZ and one sample from SFV-Nb6p-Fc were excluded from this analysis due to low sample viability. *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 7] Figure 7. Antitumor activity of a DNA / RNA layered SFV vector expressing Nb11-Fc delivered by electroporation. (A) Schematic diagram of the pBK-SFV DNA plasmid vector carrying an SFV replicon (pBK-SFV-Nb11-Fc) expressing Nb11-Fc under the control of a CMV promoter. (B) Analysis confirmed in vitro expression of Nb11-Fc from the pBK-SFV plasmid by Western blot analysis from transfected BHK-21 cells containing pBK-SFV-LacZ and pBK-SFV-Nb6p-Fc. (C) Experimental design used for delivery of the pBK-SFV plasmid to tumors. Mice bearing approximately 20 mm3 MC38 subcutaneous tumors were intratumorally injected with 20 μg of pBK-SFV plasmid at the indicated times and electroporated, as described in Materials and Methods. (D) Tumor growth curve. Data represent mean ± SEM (n = 6-7). Arrows indicate treatment days. (E) Survival curves of treated animals. (F) Survival rates after tumor rechallenge in animals that achieved complete remission after treatment. Naive, untreated mice were included as controls. *, p<0.05; ****, p<0.0001; ns, not significant. [Figure 8] Figure 8. Characterization of anti-PD-L1 nanobodies and anti-PD-1 nanobodies. (A-D) The ability of anti-PD-L1 nanobodies and anti-PD-1 nanobodies to inhibit the interaction between human and mouse PD-1 and PD-L1 was assessed by ELISA using commercially available PD-L1 ectodomains fused to human IgG1 Fc. (A) Human PD-1 / PD-L1 binding inhibition curves for various nanobodies used at 3.3 nM. A control anti-hPD-L1 monoclonal antibody (aPDL1 mAb) was used at 66 nM. (B) Human PD-1 / PD-L1 binding inhibition curves for the best nanobodies selected in (A). (C) Mouse PD-1 / PD-L1 binding inhibition curves for various nanobodies used at 300 nM. A control anti-mPD-L1 monoclonal antibody (aPDL1 mAb) was used at 130 nM. (D) Mouse PD-1 / PD-L1 binding inhibition curves for the best nanobodies selected in (C). In A–D, data represent the percentage of binding inhibition, considering 100% binding as in the absence of antibody. Ctrl Nb, control nanobody. [Figure 9] Figure 9. Antitumor activity of SFV vectors encoding nanobody-Fc fusion proteins in the B16OVA tumor model. Mice were injected with 5 x 10 B16OVA cells in the right flank. When tumors reached a size of approximately 20 mm, they were treated intratumorally with 3 x 10 viral particles of the indicated SFV vector or saline. A) Tumor growth after treatment. Data represent mean ± SEM (n = 9 / group). B) Survival curves of animals treated with the various vectors. **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 10] Figure 10. Abscopal effect induced by the SFV-Nb11-Fc vector. Mice bearing two subcutaneous contralateral MC38 tumors were treated by injecting two doses of 3 x 10 viral particles of SFV-Nb11-Fc or saline into the right flank tumor on days 0 and 5. (A) Tumor growth progression of treated tumors (solid line) or untreated tumors (dashed line). Data represent mean ± SEM (n = 6-7). B) Survival curve. *p<0.01; ****, p<0.0001; ns, not significant. [Figure 11] Figure 11. Immune cell characterization in draining lymph nodes from MC38 tumors treated with SFV vectors encoding Nb-Fc. Mice (n = 5-6 / group) bearing approximately 50 mm3 MC38 tumors were administered a single it dose of 3 x 108 SFV viral particles. Five days later, they were sacrificed and immune cell infiltration within the tumor-draining lymph nodes (dLNs) was analyzed by flow cytometry. (A) Analysis of CD8+ T cells. (B) Analysis of myeloid populations (CD11b+ cells). Asterisks above the bars indicate comparison with the saline group. One sample from SFV-LacZ and one sample from SFV-Nb11-Fc were excluded from the analysis in panel A due to poor sample viability. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 12] Figure 12. Diagram of SFV vectors expressing ICK. Diagram of SFV vectors encoding nanobodies (Nb) fused to either scIL12 (A) and dcIL12 (B), with the protein products shown on the right (not to scale). The subgenomic promoter (sgPr) enabling transcription of the subgenomic RNA encoding the Nb is indicated by the arrow. [Figure 13] Figure 13. Analysis of ICK expression. (A) BHK-21 cells were infected with SFV viral particles expressing the indicated transgenes at a multiplicity of infection (MOI) of 10 or mock infected and analyzed at 24 h by a specific mouse IL-12 ELISA for quantification. (B) To measure IL-12 and ICK activity, supernatants from cells infected with SFV-ICK viral particles were added to splenocytes from healthy mice, incubated for 48 h, and quantified by a commercially available IFNγ-specific ELISA. sc, scIL12; dc, dcIL12. [Figure 14]Figure 14. Antitumor effect of SFV vectors expressing ICK. The antitumor activity of SFV vectors expressing ICK was tested in an MC38 subcutaneous tumor model. When tumors reached a size of approximately 20 mm, a single intratumoral dose of 2 x 107 viral particles of the indicated vector was administered. Saline was used as a negative control. (A) Tumor growth progression (mean ± SEM, n = 7-8 / group). (B) Survival rate (numbers indicate the number of complete regressions / mouse). (C) Serum IL-12 levels 24 hours after treatment. (D) Body weight progression. [Figure 15] Figure 15. Dose-finding study of SFV-dcIL12-Nb11 and SFV-dcIL12. The antitumor activity of SFV-dcIL12-Nb11 and SFV-dcIL12 was compared in an MC38 subcutaneous tumor model. When tumors reached a size of approximately 20 mm3, a single intratumoral dose of the indicated vector was administered at the indicated dose. Saline was used as a negative control. (A) Tumor growth progression (mean ± SEM, n = 7 / group). (B) Survival rate (numbers indicate the number of complete regressions / mouse). *, p<0.05; **, p<0.01; ****, p<0.0001; ns, not significant. DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs at the time of filling. However, in the event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0028] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise indicated, definite articles such as "the" used herein also include the plural of the noun.

[0029] Nanobodies A first aspect of the disclosure provides a Nanobody selected from (i) an anti-PD-L1 Nanobody and (ii) an anti-PD-1 Nanobody, wherein the Nanobody comprises the following complementarity determining regions (CDRs): (i) a CDR1 described by SEQ ID NO: 1, a CDR2 described by SEQ ID NO: 2, and a CDR3 described by SEQ ID NO: 3, or (ii) a CDR1 described by SEQ ID NO: 5, a CDR2 described by SEQ ID NO: 6, and a CDR3 described by SEQ ID NO: 7.

[0030] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" are used interchangeably and refer to antibody fragments consisting of a single monomeric variable antibody domain. This is the smallest fully functional antigen-binding fragment. Structurally, nanobodies resemble the heavy chain variable domain of conventional antibodies, with three hypervariable complementarity-determining regions (CDRs) interspersed with four highly conserved regions called framework regions (FRs).

[0031] Thus, the present invention also includes fragments, derivatives and analogs of Nanobodies. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the same biological function or activity of a Nanobody of the invention. A polypeptide fragment, derivative, or analog of the invention can be (i) a polypeptide with one or more conservative or non-conservative amino acid residue substitutions, or (ii) a polypeptide with substitutions at one or more amino acid residues, or (iii) a polypeptide formed by fusing any of the above with another compound (see below for Nanobody conjugates).

[0032] A Nanobody of the invention refers to a polypeptide comprising the above-described CDR regions that has PD-L1 protein-binding activity or PD-1 protein-binding activity. For example, the present disclosure includes not only intact Nanobodies, but also active Nanobody or Nanobody conjugate fragments formed from Nanobodies with other sequences (see below for Nanobody conjugates). The term also encompasses mutant forms of polypeptides comprising the above-described CDR regions that have the same function as a Nanobody of the invention but may differ in their sequence. These modifications include, but are not limited to, deletion, insertion, and / or substitution of one or several (e.g., 1-30, 1-20, or 1-10) amino acids, as well as addition of one or several (generally fewer than 20, or fewer than 10, or fewer than 5) amino acids at the C-terminus and / or N-terminus. For example, it is well known in the art that substitution of amino acids (natural or unnatural) with similar or similar properties does not typically alter the function of a protein. In another example, addition of one or several amino acids at the C-terminus and / or N-terminus does not typically alter the function of a protein. Contemplated variations also include polypeptides having substitutions at one or more amino acid residues, as well as homologous sequences, conservative variants, allelic variants, naturally occurring variants and induced variants.

[0033] In the present disclosure, "conservative variant" refers to a polypeptide in which, compared to the amino acid sequence of a Nanobody of the invention, there are amino acids substituted by amino acids with similar or similar properties. Usually, there are up to 10, up to 8, up to 5, or most usually up to 3 substituted amino acids. These conservative variant polypeptides can be produced according to the amino acid substitutions in Table 1. [Table 1]

[0034] In one embodiment of the first aspect, the Nanobody according to the first aspect blocks the binding of PD-L1 to PD-1 with a half maximal inhibitory concentration (IC50) of 6 nM or less. The "half maximal inhibitory concentration (IC50)" is a measure of the potency of a substance in inhibiting a particular biological or biochemical function. IC50 is a quantitative measure of how much of a particular inhibitor (e.g., drug) is required to inhibit a given biological process by 50% in vitro. In the present disclosure, the biological process inhibited is the binding of PD-L1 to PD-1. IC50 can be measured, for example, by competitive ELISA, which measures the binding of PD-1 to PD-L1 in the presence of various concentrations of the Nanobody being tested. In certain embodiments, the IC50 of the Nanobody is 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less.

[0035] In one embodiment of the first aspect, the Nanobody is humanized. In another embodiment, the Nanobody is recombinant. In another embodiment, the Nanobody is isolated. The term "humanized" Nanobody refers to a Nanobody whose peptide sequence has been modified to increase its similarity to antibody variants naturally produced in humans. This can be done by replacing one or more amino acid residues in the amino acid sequence of the Nanobody sequence (particularly in the framework sequences) with one or more of the amino acid residues present at the corresponding positions in the VH domain from a conventional antibody of human origin. The term "recombinant" Nanobody refers to such molecules that are produced, expressed, isolated, or obtained by techniques or methods known in the art, for example, as recombinant DNA technology, including DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice), or cellular (e.g., CHO cell) expression systems, or non-human cellular expression systems (e.g., yeast, bacteria, insects), or antibodies isolated from a recombinant combinatorial human antibody library. The term "isolated" refers to materials that are at least partially free from other biological molecules from the cell or cell culture in which they are produced, e.g., at least partially free from expression system components such as biological molecules from the host cell or of its growth medium, or at least partially free from cellular components of the organism (e.g., animal) from which they are derived. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of pharmaceutical formulations that contain the referenced material.

[0036] In a particular embodiment of the first aspect, the Nanobody comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO: 4. In a more particular embodiment, the Nanobody comprises or consists of a sequence having at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 4. In an even more particular embodiment, the Nanobody comprises or consists of the sequence set forth in SEQ ID NO: 4.

[0037] In a particular embodiment of the first aspect, the Nanobody comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO: 8. In a more particular embodiment, the Nanobody comprises or consists of a sequence having at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 8. In an even more particular embodiment, the Nanobody consists of the sequence set forth in SEQ ID NO: 8.

[0038] In the present invention, the term "identity" refers to the percentage of residues that are identical between two sequences when the sequences are optimally aligned. In optimal alignment, if a position in a first sequence is occupied by the same amino acid residue as the corresponding position in a second sequence, the sequences are considered to be identical at that position. The percentage of identity determines the number of identical residues over a specified length in a given alignment. Therefore, the level of identity between two sequences, or "percent sequence identity," is measured as the ratio of the number of identical positions shared by the sequences to the number of positions compared (i.e., percent sequence identity = (number of identical positions / total number of positions compared) x 100). Gaps, i.e., positions in the alignment where a residue exists in one sequence but not in the other, are considered to be positions with non-identical residues and are counted as comparison positions.

[0039] Several mathematical algorithms for rapidly obtaining optimal alignments and calculating percent identity between two or more sequences are known and are incorporated into several available software programs. For the purposes of the present invention, sequence identity between two amino acid sequences is determined using an algorithm based on global alignment, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970. DOI:10.1016 / 0022-2836(70)90057-4), preferably implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet. 16:276-277, 2000. DOI:10.1016 / s0168-9525(00)02024-2), or the BLAST Global Alignment tool (Altschul et al., "Basic local alignment search tool", 1990, J. Mol. Biol., v. 215, pages 215-220) using default settings. 403-410, 1990. DOI:10.1016 / S0022-2836(05)80360-2). Local alignments can also be used if the sequences being compared are substantially the same length.

[0040] Nanobody conjugates As mentioned above, the second aspect relates to a nanobody conjugate comprising a nanobody according to the first aspect.

[0041] Nanobodies can be readily conjugated to other molecules, such as other proteins or effector domains, to form nanobody conjugates with tailored utility for specific therapeutic applications. The term "nanobody conjugate" refers to a nanobody as defined herein conjugated (bound) to at least a second molecule. The nanobody and additional molecule are operatively associated within the conjugate. Embodiments of this aspect relate to nanobody conjugates comprising, in addition to one or more nanobodies according to the first aspect, a molecule selected from the group consisting of albumin, a detectable marker, a radionuclide, a drug, a toxin, a polymer, a purification tag, a liposome, a nanoparticle, and combinations thereof. Nanobodies can be conjugated (or fused) to other molecules via the amino or carboxy terminus. In some embodiments, nanobodies are conjugated to one or more molecules via both the amino and carboxy termini. All of the embodiments defined above for nanobodies of the first aspect also apply to nanobody conjugates of the second aspect.

[0042] In some embodiments, the nanobody conjugate is monospecific. In some embodiments, the nanobody conjugate is bispecific. In other embodiments, the nanobody conjugate is multispecific. As used herein, monospecific, bispecific, or multispecific refers to having affinity for one, two, or more antigens, respectively. The specificity can be provided by one or more nanobodies or other molecules capable of specifically binding to a particular target.

[0043] Immunoconjugates according to the present disclosure also contemplate a nanobody conjugated to at least another nanobody (naked nanobody). The nanobody and other molecule can be linked directly or via a linker (or spacer). As used herein, a "linker" is a peptide between 1 and 50 amino acids in length, typically selected or designed to be unstructured and flexible. These include, but are not limited to, synthetic peptides rich in Gly, Ser, Thr, Gln, Glu, or additional amino acids frequently associated with unstructured regions in natural proteins. A suitable linker is, for example, a flexible glycine-serine linker.

[0044] In one embodiment, the nanobody conjugate according to the second aspect comprises a nanobody as defined above and at least one further nanobody. In one embodiment, the nanobodies are conjugated in tandem, typically via a linker. For example, in one embodiment, tandem repeats of two, three, four, five or more nanobodies fused to each other directly or via a linker are disclosed. Configurations other than tandem are also possible. To increase the in vivo half-life of the nanobody conjugate, the nanobody may be linked to another molecule, such as albumin.

[0045] In another embodiment, the immunoconjugate comprises, in addition to the nanobody defined above, an antibody or antibody fragment, such as an antibody Fc fragment or an antibody scFv fragment. In a specific embodiment, the nanobody conjugate comprises an immunoglobulin Fc domain. The "Fc domain" or "fragment crystallizable domain" is the tail region of an antibody, which interacts with cell surface receptors called Fc receptors and with some proteins of the complement system. In a more specific embodiment, the Fc domain is selected from the group consisting of an IgG Fc domain, an IgM Fc domain, an IgA Fc domain, and an IgE Fc domain, and more specifically an IgG Fc domain. In a specific embodiment, the nanobody conjugate comprises two nanobodies bound to an immunoglobulin Fc domain (Fc dimer). The nanobodies within the Fc dimer can be the same (Fc homodimer) or different (Fc heterodimer). In a further specific embodiment, the nanobody conjugate is an Fc homodimer. In a specific embodiment, the Fc domain is a murine IgG domain. In another specific embodiment, the Fc domain is a human IgG domain. In another specific embodiment, the Fc domain is a humanized IgG domain. As shown in the examples below, Fc dimers comprising an anti-PD-L1 nanobody and an anti-PD1 nanobody reduced the PD-1 / PD-L1 50% inhibitory concentration (IC50) 8-fold and 40-fold, respectively, and further generated highly potent anti-tumor responses in a murine colon adenocarcinoma MC38 model, resulting in >50% complete regressions and improved therapeutic efficacy compared to conventional mAbs. These effects were also observed in a murine B16-OVA melanoma model. Treated animals that achieved complete remission remained tumor-free after rechallenge with MC38 cells (Figure 4, right panel), suggesting that these treatments were able to generate an efficient memory immune response. An exemplary schematic of an Fc dimer can be seen in Figure 2A.

[0046] In a particular embodiment of the first aspect, the Nanobody conjugate comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO: 9. In a more particular embodiment, the Nanobody conjugate comprises or consists of a sequence having at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 9. In an even more particular embodiment, the Nanobody conjugate comprises or consists of the sequence set forth in SEQ ID NO: 9.

[0047] In a particular embodiment of the first aspect, the Nanobody conjugate comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% sequence identity to SEQ ID NO: 10. In a more particular embodiment, the Nanobody conjugate comprises or consists of a sequence having at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 10. In an even more particular embodiment, the Nanobody conjugate comprises or consists of the sequence set forth in SEQ ID NO: 10.

[0048] In another embodiment, the nanobody conjugate is a chimeric antigen receptor (CAR). In certain embodiments, the CAR is encoded by a vector as defined above. An expression vector containing a CAR comprising the above-mentioned nanobody can be useful for preparing CAR T cells (chimeric antigen receptor T cells). As known in the art, CAR T cells are T cells genetically engineered to produce an artificial T cell receptor. Thus, the present disclosure contemplates CAR T cells, whose CAR comprises the above-mentioned nanobody. CAR T cells comprising multiple identical or different nanobodies are also contemplated.

[0049] In another embodiment, the nanobody conjugate according to the second aspect comprises a nanobody and an effector molecule. As used herein, "effector molecule" refers to a molecule that selectively binds to a protein and modulates its biological activity.

[0050] In certain embodiments, the nanobody conjugate comprises a cytokine. These nanobody conjugates are also referred to herein as "immunocytokines" or "ICKs." The cytokine may be selected from the group consisting of IL-12, IL-2, IL-15, IL-18, IL-21, IL-33, IL-7, IFN-gamma, IFN-alpha, and IFN-beta. Particularly desirable cytokines are cytokines with anti-tumor activity, such as IL-12. The nanobody may be fused to the amino or carboxy terminus of the cytokine, or to both the amino and carboxy termini of the cytokine, either directly or via a linker. In certain embodiments, the immunocytokine comprises a single-chain version of IL-12. In another specific embodiment, the immunocytokine comprises a two-chain version of IL-12. An exemplary schematic diagram of an immunocytokine according to the present disclosure can be seen in Figure 12. The advantages of these immunocytokines are related to (i) a synergistic effect on the action of the cytokine and the anti-PD-1 or anti-PD-L1 nanobody, (ii) a superior activity of the cytokine within the tumor due to the fact that high levels of PD-L1 and PD-1 expression within the tumor allow the nanobody-mediated retention of ICK within the tumor, (iii) a reduction in the potential toxicity of cytokines retained within the tumor tissue, and (iv) an increased stability of the nanobody due to fusion to the cytokine.

[0051] In some particular embodiments of the second aspect, the Nanobody conjugate comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, more particularly at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

[0052] In particular embodiments, the nanobody conjugate comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. In another very particular embodiment, the nanobody conjugate is a dimer comprising or consisting of SEQ ID NO: 15 and SEQ ID NO: 16. In another very particular embodiment, the nanobody conjugate is a dimer comprising or consisting of SEQ ID NO: 15 and SEQ ID NO: 18. In another very particular embodiment, the nanobody conjugate is a dimer comprising or consisting of SEQ ID NO: 17 and SEQ ID NO: 16. In another very particular embodiment, the nanobody conjugate is a dimer comprising or consisting of SEQ ID NO: 17 and SEQ ID NO: 18.

[0053] In another specific embodiment, the nanobody conjugate comprises a polymer. A non-limiting polymer contemplated in this embodiment is a polymer that increases the half-life of the polypeptide, for example, polyethylene glycol.

[0054] In another specific embodiment, the nanobody conjugate comprises a purification tag, for example a His tag.

[0055] In another specific embodiment, the nanobody conjugate comprises a detectable marker, such as a radioactive molecule, a fluorescent molecule, a chromogenic molecule, or an enzyme. In another embodiment, the nanobody conjugate comprises a nanoparticle, such as a gold nanoparticle, a quantum dot, or a magnetic nanoparticle. In another embodiment, the nanobody conjugate comprises a liposome or a vesicle. In another embodiment, the nanobody conjugate comprises a drug, such as a chemotherapeutic agent.

[0056] Polynucleotides, Vectors, Viral Particles and Recombinant Technology The Nanobodies and Nanobody conjugates disclosed herein may be administered directly, i.e., in protein form, or as a polynucleotide encoding the polypeptide, which is expressed by recipient cells in vitro or in vivo. This means that the polynucleotide is delivered intracellularly, for example, by intratumoral administration of a DNA or RNA vector encoding the Nanobody or Nanobody conjugate, whereby the host cells produce the Nanobody or Nanobody conjugate in situ.

[0057] The third aspect therefore relates to a polynucleotide encoding a nanobody or nanobody conjugate as defined above. Any of the embodiments defined above for a nanobody or nanobody conjugate also apply to the third aspect. The term "polynucleotide encoding a nanobody or nanobody conjugate" includes a polynucleotide that encodes a nanobody or nanobody conjugate and may also contain additional coding and / or non-coding sequences. The polynucleotide of the third aspect may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

[0058] In one embodiment of the third aspect, the polynucleotide comprises a sequence encoding a CDR1 described by SEQ ID NO:1, a sequence encoding a CDR2 described by SEQ ID NO:2, and a sequence encoding a CDR3 described by SEQ ID NO:3.

[0059] In another embodiment of the third aspect, the polynucleotide comprises a sequence encoding a CDR1 described by SEQ ID NO:5, a sequence encoding a CDR2 described by SEQ ID NO:6, and a sequence encoding a CDR3 described by SEQ ID NO:7.

[0060] In a particular embodiment of the third aspect, the polynucleotide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, more particularly at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 19. In an even more particular embodiment, the polynucleotide has the sequence set forth in SEQ ID NO: 19.

[0061] In a particular embodiment of the third aspect, the polynucleotide comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, more particularly at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 20. In an even more particular embodiment, the polynucleotide has the sequence set forth in SEQ ID NO: 20.

[0062] Full-length nucleotide sequences encoding the Nanobodies or Nanobody conjugates of the present disclosure can generally be obtained by PCR amplification or / and recombinant methods. Once a polynucleotide sequence is obtained, recombinant methods can be used to obtain the relevant sequence on a large scale. Typically, the sequence can be obtained by cloning it into a vector, introducing it into cells, and then isolating the sequence from the host cells grown by conventional methods. Polynucleotide sequences encoding the Nanobodies or Nanobody conjugates of the present disclosure can also be obtained by chemical synthesis.

[0063] The DNA sequence can then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art. Thus, the present invention also relates in a fourth aspect to a vector comprising the above-mentioned polynucleotide of the third aspect. In particular embodiments of this fourth aspect, the vector is an expression vector containing a suitable promoter and optionally also regulatory sequences. These vectors can be used to express Nanobodies or Nanobody conjugates in vitro or in vivo.

[0064] Non-limiting examples of suitable expression vectors within the meaning of the present disclosure may be derived from viruses such as alphaviruses, adenoviruses, herpesviruses, lentiviruses, retroviruses, poxviruses, and Newcastle disease viruses. In one embodiment, the expression vector is derived from an alphavirus, such as Sindbis virus, Venezuelan equine encephalitis virus (VEEV), or Semliki Forest virus (SFV). In a specific embodiment, the expression vector is an SFV vector. In even more specific embodiments, the expression vector is a replication-deficient alphavirus vector based on Semliki Forest virus (SFV). In even more specific embodiments, the expression vector is based on a replication-competent SFV vector that expresses viral structural proteins. In even more specific embodiments, the expression vector is based on a replication-incompetent SFV vector from which viral structural proteins have been partially or completely eliminated. In some embodiments, the expression vector comprises a polynucleotide of interest according to the third aspect, located under the control of a subgenomic promoter (sgPr). In a specific embodiment, the polynucleotide of interest is under the control of a subgenomic promoter (sgPr) fused to the SFV capsid translation enhancer via the 2A autoprotease of the foot-and-mouth disease virus. As already mentioned, SFV vectors have been shown to successfully deliver the nanobodies and nanobody conjugates defined above. As shown in the examples below, delivery of the nanobodies described herein via SFV vectors not only enabled high local intratumoral expression of the nanobodies or nanobody conjugates, but also enhanced their intratumoral activity.

[0065] In some particular embodiments of the fourth aspect, the vector comprises or consists of a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, more particularly at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In even more particular embodiments, the vector comprises or consists of a sequence selected from SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

[0066] The vector according to the fourth aspect can be used to produce SFV viral particles that can efficiently infect tumor cells and express the encoded nanobody or nanobody conjugate in vivo. Viral particles are small particles that contain specific proteins from the viral envelope but little or no viral genetic material and are unable to cause infection. Therefore, the present disclosure also contemplates viral particles comprising a vector according to the fourth aspect, as well as methods for producing the same. To produce viral particles, a vector comprising a polynucleotide encoding a nanobody or nanobody conjugate, along with polynucleotides encoding specific viral proteins such as viral capsid and envelope proteins, is delivered to host cells. The cells are then cultured under conditions that produce viral particles, which are then harvested and purified by conventional means.

[0067] In one embodiment, the method of producing viral particles comprises: (i) transcribing a vector comprising a polynucleotide encoding the Nanobody or Nanobody conjugate; (ii) delivering the RNA to the host cell; (iii) delivery of helper RNA encoding capsid and envelope proteins to the host cell; (iv) culturing the host cells under conditions appropriate for producing viral particles; (v) harvesting the culture supernatant; and (vi) Harvesting viral particles from the supernatant.

[0068] In a specific embodiment, the vector containing the polynucleotide encoding the nanobody or nanobody conjugate is an SFV vector as defined above. In another specific embodiment, the helper RNA is SFV-helper C-S219A and SFV-helper S2. Transcription can be carried out by conventional methods. For example, transcription from a viral vector can be carried out by m 7 This can be achieved using SP6 RNA polymerase in the presence of a G(')ppp(5')G RNA Cap Structure Analog. Delivery of RNA into host cells can also be achieved by conventional transformation methods, such as electroporation, microinjection, liposome packaging, calcium phosphate co-precipitation, etc. In certain embodiments, the RNA encoding the Nanobody or Nanobody conjugate and the helper RNA are co-transformed into the host cell.

[0069] In another specific embodiment, the host cell for producing viral particles is a eukaryotic cell, particulate, higher eukaryotic cell, more specifically, a mammalian cell selected from, for example, CHO-K1, BHK, Vero, and Vero E6, and even more specifically, a BHK cell, such as BHK-21. Depending on the host cell used, the medium used for culturing can be selected from various conventional media. The culturing is carried out under conditions suitable for host cell growth. The harvesting of the supernatant and the collection of the viral particles contained therein can also be carried out by conventional methods. For example, the harvesting can be carried out by centrifugation, and the viral particles can be collected by ultracentrifugation.

[0070] The present disclosure also relates to viral particles obtainable or obtained by the above methods.

[0071] As mentioned above, the vector of the fourth aspect or the host cell of the fifth aspect, comprising a polynucleotide encoding a Nanobody or Nanobody conjugate as defined above, can also be used to recombinantly produce the Nanobody or Nanobody conjugate in vitro, which may then be delivered to tumor cells as a recombinant product. Accordingly, a seventh aspect provides a method of producing a Nanobody or Nanobody conjugate as defined above, comprising the steps of: (a) culturing a host cell comprising an expression vector as defined above that encodes the Nanobody or Nanobody conjugate under conditions suitable for producing the Nanobody or Nanobody conjugate, thereby obtaining a culture containing the Nanobody or Nanobody conjugate; and (b) isolating or recovering the Nanobody or Nanobody conjugate from the culture.

[0072] Generally, host cells for in vitro expression of Nanobodies or Nanobody conjugates can be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples include bacterial cells, such as Escherichia coli, Streptomyces, or Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 or Sf9; and animal cells, such as BHK cells, CHO cells, Vero cells, Vero E6 cells, COS7 cells, and 293 cells. In one embodiment of the seventh aspect, the host cell is Escherichia coli. In another embodiment, the host cell is selected from Saccharomyces cerevisiae and Pichia pastoris. In another particular embodiment, the host cell is a mammalian cell selected from, for example, BHK, CHO-K1, Vero, and Vero E6, more particularly a BHK cell such as BHK-21.

[0073] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method. The procedure used is well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation, if necessary. When the host is a eukaryotic organism, transfection can be carried out by electroporation, microinjection, liposome packaging, calcium phosphate co-precipitation, etc. The resulting transformant can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention. As already mentioned above, depending on the host cell used, the medium used for cultivation can be selected from various conventional media, and the cultivation is carried out under conditions suitable for host cell growth. After the host cells have been grown to an appropriate cell density, the selected promoter can be induced by an appropriate method (such as temperature shift or chemical induction), and the cells are incubated for an additional period.

[0074] The recombinant polypeptide in the above method may be expressed intracellularly, expressed on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods, taking advantage of its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic disruption, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorbent bed analysis, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations thereof.

[0075] The present disclosure also contemplates a recombinant Nanobody or recombinant Nanobody conjugate obtainable or obtainable by the above method.

[0076] Nanobodies or nanobody conjugates can also be delivered to cells in vivo by non-viral methods, for example, by delivering RNA or DNA vectors to target cells without being vehiculated via viral particles. As shown in the examples below, this last option was also successfully used to deliver the dimeric nanobodies defined above to tumor cells, resulting in antitumor effects comparable to those obtained with viral particles. An additional advantage of the DNA system is that neutralizing antibodies against SFV are not induced, allowing for multiple administrations. The nanobodies or nanobody conjugates defined above can also be delivered as RNA or DNA using a plasmid in which the SFV vector sequence is placed under a eukaryotic promoter, such as a CMV promoter. In this DNA / RNA system, SFV RNA is transcribed in the transfected cells, resulting in high transgene expression levels and apoptosis similar to those in SFV-infected cells.

[0077] composition An eighth aspect of the present disclosure relates to a pharmaceutical composition comprising a Nanobody, or Nanobody conjugate, or expression vector, or host cell, or viral particle, all as defined above, together with a pharmaceutically acceptable excipient and / or carrier. Any of the embodiments described above for a Nanobody, a Nanobody conjugate, an expression vector, a host cell, or a viral particle also apply to the pharmaceutical composition. The phrase "pharmaceutically acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other components of the pharmaceutical composition. It must also be suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0078] The choice of pharmaceutical formulation depends on the properties of the active compound and its route of administration. Any route of administration can be used. In some embodiments, the route of administration is parenteral, in which case the composition is suitable for parenteral administration. In certain embodiments, the route of administration is by injection. In more particular embodiments, the route of administration is systemic, for example, by intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, or transdermal injection. In certain embodiments, the route of administration is local, for example, by intratumoral injection. The intratumoral administration route is particularly suitable for delivery of the nanobodies or nanobody conjugates of the invention by vectors, for example, viral vectors, or by viral nanoparticles, although the nanobodies or nanobody conjugates of the invention can also be administered by this route in protein form. Topical administration is also contemplated, as can the pharmaceutical composition be a topical composition.

[0079] The pharmaceutical compositions may be in any form, including tablets, pellets, capsules, aqueous or oily solutions, suspensions, emulsions, aerosols or dry powder forms suitable for reconstitution with water or other suitable liquid medium before use, for immediate or delayed release, among others.

[0080] Suitable excipients and / or carriers and their amounts can be easily determined by those skilled in the art according to the type of preparation to be prepared.Examples of suitable pharmaceutically acceptable excipients include solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Use of any conventional excipient medium is considered within the scope of the present invention, except that it is incompatible with the substance or its derivatives, for example, by producing any undesirable biological effects or otherwise interacting in a harmful manner with any other components of the pharmaceutical composition.

[0081] The term "carrier" is understood as a pharmaceutically acceptable vehicle. Carriers can be organic, inorganic, or both. Suitable carriers well known to those skilled in the art include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Carriers can also include saline, buffer solutions, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, carriers can be polycationic polymers, vesicles, liposomes, or nanoparticles.

[0082] Typically, pharmaceutical compositions contain a therapeutically effective amount of a Nanobody, Nanobody conjugate, expression vector, host cell, or viral particle. As used herein, the phrase "therapeutically effective amount" refers to an amount of a compound that, when administered, is sufficient to prevent the onset of one or more symptoms of the disease being addressed or to alleviate to some extent one or more symptoms of the disease being addressed. The specific dose of a compound administered in accordance with the present disclosure will, of course, be determined by the particular circumstances surrounding the case, including the compound being administered, the route of administration, the specific condition being treated, and similar considerations. A therapeutically effective amount may be 0.001 to 50% by weight, particularly 0.01 to 10% by weight, and more preferably 0.1 to 5% by weight of the Nanobody, Nanobody conjugate, expression vector, host cell, or viral particle. The amount (or dose) of the active ingredient administered can be from about 10 micrograms / kilogram body weight to about 50 milligrams / kilogram body weight per day, particularly the dose can be from about 100 micrograms / kilogram body weight to about 40 milligrams / kilogram body weight, and more particularly the dose can be from about 1 milligram / kilogram body weight to about 30 milligrams / kilogram body weight. In addition, the nanobodies, nanobody conjugates, vectors, or viral particles of the disclosure may also be used in conjunction with other therapeutic agents.

[0083] The present disclosure also contemplates a diagnostic composition comprising a nanobody or nanobody conjugate as defined above.

[0084] Also disclosed herein is a kit of parts comprising: (a) a nanobody, nanobody conjugate, vector or viral particle as defined above, optionally together with a pharmaceutically acceptable excipient or carrier; (b) optionally, an additional therapeutic agent, and (c) optionally, instructions for its use.

[0085] Also disclosed herein is a container or injection device comprising a nanobody, nanobody conjugate, vector or viral particle as defined above, preferably together with a pharmaceutically acceptable excipient or carrier.

[0086] The present disclosure also contemplates a kit of parts including: (a) a nanobody or nanobody conjugate as defined above, (b) optionally, additional means for performing the diagnosis (e.g., buffers, reagents, controls), and (c) optionally, instructions for its use.

[0087] use In a ninth aspect, the present disclosure provides a pharmaceutical composition, or a nanobody conjugate, or an expression vector, or a host cell, or a viral particle, or a kit-of-parts, any of which are defined above, for use in therapy. In certain embodiments, the therapy is immunotherapy. In other embodiments, the therapy comprises blocking or inhibiting the binding of human PD-L1 to human PD-1. The present invention also contemplates the nanobody, nanobody conjugate, expression vector, host cell, viral particle, or pharmaceutical composition for use as an immune checkpoint inhibitor.

[0088] A tenth aspect provides a Nanobody, or Nanobody conjugate, or expression vector, or host cell, or viral particle, or pharmaceutical composition, or kit-of-parts, any of which are defined above, for use in the treatment of cancer. In other words, the tenth aspect provides a pharmaceutical composition, or Nanobody, or Nanobody conjugate, or expression vector, or host cell, or viral particle, or kit-of-parts, any of which are defined above, for use in the treatment of tumors. As used herein, the terms "cancer" or "tumor" are used interchangeably and refer to a malignant abnormal cell growth that has the potential to invade or spread to other parts of the body, as commonly understood in the art.

[0089] "Treating cancer" within the meaning of this disclosure includes prophylactic treatment before the clinical onset of cancer, or therapeutic treatment after the clinical onset of cancer, which may be achieved by preventing the onset of cancer or reversing the symptoms of cancer.

[0090] In certain embodiments of the tenth aspect, the nanobody, nanobody conjugate, expression vector, host cell, viral particle, kit of parts, or pharmaceutical composition is for use in combination therapy with surgery, radiation, or an additional therapeutic agent to treat cancer. The nanobody, nanobody conjugate, expression vector, host cell, viral particle, kit of parts, or pharmaceutical composition and the surgery, radiation, or additional therapeutic agent can be administered sequentially, simultaneously, or within a therapeutic interval. In certain embodiments, the combination therapy includes surgery. In another specific embodiment, the combination therapy includes radiation. In certain embodiments, the combination therapy includes an anti-tumor agent. The anti-tumor agent can be a cytotoxic agent, such as an alkylating agent that improves specificity, a folate antagonist, antimetabolites including purine and pyrimidine analogs, antibiotics and other natural products including anthracyclines and vinca alkaloids, and antibodies. The anti-tumor agent can also be a hormonal agent or a signal transduction inhibitor. In certain embodiments, the combination therapy includes chemotherapy. The combination therapy can also include a different immunotherapy. For example, in certain embodiments, the combination may include activated natural killer cells, (CAR) T cells, tumor-infiltrating lymphocytes, or tumor antigen-loaded dendritic cells.

[0091] The cancers that can be treated according to the tenth embodiment are not particularly limited. Non-limiting types of tumors or cancers that can be treated in the sense of this specification are carcinomas, sarcomas, and blood tumors. For example, the cancers to be treated can be carcinomas, such as adenocarcinoma, sarcoma, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, mesothelioma, fibrosarcoma, angiosarcoma, liposarcoma, glioma, myxosarcoma, mesenchymous, blood cancers, such as myeloma, leukemia, or lymphoma, and mixed tumors, such as adenosquamous carcinoma, mixed mesodermal tumor, carcinosarcoma, or teratocarcinoma. Carcinomas are predominant cancers, and are cancers of epithelial cells or cells covering the outer or inner surface of organs, glands, or other body structures (e.g., skin, uterus, lung, breast, prostate, stomach, intestine), and tend to metastasize. Carcinomas can be, for example, adenocarcinomas of the breast, lung, colon, prostate, or bladder, and squamous cell carcinoma. Sarcomas originate from connective tissue or supporting tissue (e.g., bone, cartilage, tendon, ligament, fat and muscle). Sarcomas can be osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (membranous lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (fatty tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), or mesenchymal or mixed mesodermal tumor (mixed connective tissue type). Blood tumors originate from bone marrow and lymphatic tissue. Hematologic tumors can be myelomas, which originate in the plasma cells of the bone marrow; leukemias, which are "liquid tumors," and are tumors of the bone marrow and can be myeloid or granulocytic leukemias (myeloid and granulocytic leukocytes), lymphocytic, lymphocytic, or lymphoblastic leukemias (lymphoid and lymphocytic blood cells), or polycythemia vera or erythremia (various blood cell products, but predominantly red blood cells); or solid tumors, such as lymphomas, which arise in the glands or lymph nodes of the lymphatic system and can be Hodgkin's lymphoma or non-Hodgkin's lymphoma. In addition, mixed-type tumors, such as adenosquamous carcinomas, mixed mesodermal tumors, carcinosarcoma, or teratocarcinoma, also exist. All of these types of tumors, as well as others that would be apparent to one skilled in the art, are contemplated within the meaning of this disclosure.

[0092] Tumors that can be treated in the sense of the present disclosure can also be named based on the organ from which they originate, i.e., "primary site," such as tumors or cancers of the breast, brain, lung, liver, skin, prostate, testicle, bladder, colon and rectum, cervix, uterus, blood, lymph, etc. This nomenclature often persists even if the tumor metastasizes to another part of the body different from the primary site. In this sense, the cancers contemplated in the tenth aspect can be, but are not limited to, cancers of the gastrointestinal tract, respiratory system, nervous system, hematopoietic system, epithelium, vascular system, reproductive system (such as cervix, ovary, uterus, vagina and vulva), urinary tract, endocrine system, skin, heart, brain, eye, testicle, muscle, bone, or breast.

[0093] In certain embodiments, the cancer to be treated is selected from colon adenocarcinoma and melanoma.

[0094] The eleventh to thirteenth aspects of the present disclosure relate to the use of a nanobody, nanobody conjugate or kit of parts as defined above in an assay (based on the detection of human PD-L1 and / or human PD-1 molecules) or in a diagnosis. In most embodiments of these aspects, the nanobody or nanobody conjugate is linked to a detectable molecule.

[0095] Any of the embodiments described above for the nanobodies, nanobody conjugates, expression vectors, host cells, viral particles, pharmaceutical compositions or kits of parts also apply to the therapeutic uses described in this section.

[0096] For the sake of completeness, the present specification also discloses the following numbered embodiments: 1. A single domain antibody (VHH or nanobody) comprising the following complementarity determining regions (CDRs): (i) CDR1 described by SEQ ID NO:1, CDR2 described by SEQ ID NO:2, and CDR3 described by SEQ ID NO:3.

[0097] 2. (i) an anti-PD-L1 Nanobody comprising a CDR1 set forth in SEQ ID NO: 1, a CDR2 set forth in SEQ ID NO: 2, and a CDR3 set forth in SEQ ID NO: 3, wherein the nanobody is capable of inhibiting the binding of PD-L1 to PD-1; A nanobody according to embodiment 1.

[0098] 3. A nanobody according to any one of embodiments 1-2, which blocks the binding of PD-L1 to PD-1 with a median inhibitory concentration (IC50) of 3.6 nM or less.

[0099] 4. A nanobody according to any one of embodiments 1 to 3, which inhibits the interaction of PD-1 with PD-L1 for its human and mouse orthologues.

[0100] 5. (i) the sequence set forth in SEQ ID NO: 4, or a sequence having at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% identity to SEQ ID NO: 4 5. The nanobody according to any one of embodiments 1 to 4, comprising or consisting of:

[0101] 6. A Nanobody according to the preceding embodiments, comprising or consisting of a conservative variant of SEQ ID NO: 4.

[0102] 7. A nanobody conjugate comprising a nanobody according to any one of embodiments 1 to 6.

[0103] 8. A nanobody conjugate according to the preceding embodiments, comprising 2, 3, 4, 5 or 6 nanobodies.

[0104] 9. A nanobody conjugate according to the preceding embodiment, which is monospecific.

[0105] 10. The nanobody conjugate according to embodiment 8, which is at least bispecific.

[0106] 11. A nanobody conjugate according to any one of embodiments 7 to 10, comprising at least one nanobody according to any one of embodiments 1 to 6 and an effector molecule.

[0107] 12. A nanobody conjugate according to any one of embodiments 7 to 11, comprising two nanobodies as defined in any one of embodiments 1 to 6, fused to an immunoglobulin Fc domain.

[0108] 13. A nanobody conjugate according to the preceding embodiments, comprising or consisting of SEQ ID NO: 9, or a sequence having at least 90%, at least 95%, at least 96%, at least 96%, at least 98% or at least 99% sequence identity to SEQ ID NO: 9.

[0109] 14. A nanobody conjugate according to any one of embodiments 7 to 11, comprising at least one nanobody according to any one of embodiments 1 to 6 and a cytokine.

[0110] 15. The nanobody conjugate according to the preceding embodiment, wherein the cytokine is selected from the group consisting of IL-12, IL-2, IL-15, IL-18, IL-21, IL-33, IL-7, IFN-gamma, IFN-alpha and IFN-beta.

[0111] 16. The nanobody conjugate according to the preceding embodiment, wherein the cytokine is IL-12.

[0112] 17. A nanobody conjugate according to the preceding embodiments, comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:16, or a sequence having at least 90%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:16.

[0113] 18. A nanobody conjugate according to the preceding embodiments, which is a dimer comprising, or consisting of, SEQ ID NO:15 and SEQ ID NO:16, a dimer comprising, or consisting of, SEQ ID NO:15 and SEQ ID NO:18, or a dimer comprising, or consisting of, SEQ ID NO:17 and SEQ ID NO:16.

[0114] 19. The nanobody conjugate according to any one of embodiments 7-18, which blocks the binding of PD-L1 to PD-1 with a 50% inhibitory concentration (IC50) of 19.3 nM or less.

[0115] The nanobody conjugate according to the preceding embodiments, which blocks the binding of PD-L1 to PD-1 with a 50% inhibitory concentration (IC50) of 20.1 nM or less, particularly 0.5 nM or less, and more particularly 0.1 nM or less.

[0116] 21. A single domain antibody (VHH or nanobody) comprising the following complementarity determining regions (CDRs): CDR1 described by SEQ ID NO:5, CDR2 described by SEQ ID NO:6, and CDR3 described by SEQ ID NO:7.

[0117] 22. An anti-PD-1 Nanobody according to embodiment 21, comprising a CDR1 described by SEQ ID NO: 5, a CDR2 described by SEQ ID NO: 6, and a CDR3 described by SEQ ID NO: 7, wherein the Nanobody is capable of inhibiting the binding of PD-L1 to PD-1.

[0118] 23. The nanobody according to any one of embodiments 21-22, which blocks the binding of PD-L1 to PD-1 with a median inhibitory concentration (IC50) of 23.6 nM or less.

[0119] 24. The nanobody according to any one of embodiments 21-23, which inhibits the interaction of PD-1 with PD-L1 for its human and mouse orthologues.

[0120] 25. The sequence set forth in SEQ ID NO: 8 or a sequence having at least 90%, or at least 95%, or at least 98%, or at least 99% identity to SEQ ID NO: 8 25. The nanobody according to any one of embodiments 21 to 24, comprising or consisting of:

[0121] 26. A Nanobody according to the preceding embodiment, comprising or consisting of a conservative variant of SEQ ID NO: 8.

[0122] 27. A nanobody conjugate comprising a nanobody according to any one of embodiments 21 to 26.

[0123] 28. A nanobody conjugate according to the preceding embodiment, comprising 2, 3, 4, 5 or 6 nanobodies.

[0124] 29. A nanobody conjugate according to the preceding embodiment, which is monospecific.

[0125] 30. The nanobody conjugate according to embodiment 28, which is at least bispecific.

[0126] 31. A nanobody conjugate according to any one of embodiments 27 to 30, comprising at least one nanobody according to any one of embodiments 20 to 26 and an effector molecule.

[0127] 32. A nanobody conjugate according to any one of embodiments 27 to 31, comprising two nanobodies as defined in any one of embodiments 21 to 26, fused to an immunoglobulin Fc domain.

[0128] 33. A nanobody conjugate according to the preceding embodiments, comprising or consisting of SEQ ID NO: 10, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 10.

[0129] 34. A nanobody conjugate according to any one of embodiments 27 to 31, comprising at least one nanobody according to any one of embodiments 21 to 26 and a cytokine.

[0130] 35. The nanobody conjugate according to the preceding embodiment, wherein the cytokine is selected from the group consisting of IL-12, IL-2, IL-15, IL-18, IL-21, IL-33, IL-7, IFN-gamma, IFN-alpha and IFN-beta.

[0131] 36. The nanobody conjugate according to the preceding embodiment, wherein the cytokine is IL-12.

[0132] 37. A nanobody conjugate according to the preceding embodiments, comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:18, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:18.

[0133] 38. A nanobody conjugate according to the preceding embodiments, which is a dimer comprising or consisting of SEQ ID NO:17 and SEQ ID NO:18, a dimer comprising or consisting of SEQ ID NO:15 and SEQ ID NO:18, or a dimer comprising or consisting of SEQ ID NO:17 and SEQ ID NO:16.

[0134] 39. The nanobody conjugate according to any one of embodiments 27-38, which blocks the binding of PD-L1 to PD-1 with a 50% inhibitory concentration (IC50) of 39.3 nM or less.

[0135] The nanobody conjugate according to the preceding embodiments, which blocks the binding of PD-L1 to PD-1 with a 50% inhibitory concentration (IC50) of 40.1 nM or less, particularly 0.5 nM or less, and more particularly 0.1 nM or less.

[0136] 41. A nanobody according to any one of embodiments 1 to 6, or 21 to 26, or a nanobody conjugate according to any one of embodiments 7 to 20, or 27 to 40, which is humanized.

[0137] 42. A polynucleotide encoding a Nanobody as defined in any one of embodiments 1 to 6, or 21 to 26, or encoding a Nanobody conjugate as defined in any one of embodiments 7 to 20, or 27 to 40.

[0138] 43. A polynucleotide according to the preceding embodiment, comprising or consisting of SEQ ID NO:19, or at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to sequence SEQ ID NO:19.

[0139] 44. A polynucleotide according to the preceding embodiment, comprising or consisting of SEQ ID NO:20, or at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to sequence SEQ ID NO:20.

[0140] 45. An expression vector comprising a polynucleotide as defined in any one of embodiments 42 to 44.

[0141] 46. ​​An expression vector according to the preceding embodiment, wherein expression is derived from an alphavirus or an adenovirus.

[0142] 47. An expression vector according to the preceding embodiment, derived from an alphavirus selected from Sindbis virus, Venezuelan equine encephalitis virus (VEEV), or Semliki Forest virus (SFV).

[0143] 48. An expression vector according to the preceding embodiment, wherein the vector is derived from SFV, in particular wherein the vector is a replication-deficient alphavirus vector based on SFV.

[0144] 49. An expression vector according to the preceding embodiments, comprising or consisting of a sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of these sequences.

[0145] 50. A host cell comprising a polynucleotide as defined in any one of embodiments 42 to 44, or an expression vector as defined in any one of embodiments 45 to 49.

[0146] 51. A host cell according to the preceding embodiments, selected from the group consisting of CHO-K1 cells, BHK cells, Vero cells and Vero E6 cells, in particular BHK cells.

[0147] 52. A viral particle comprising an expression vector according to any one of embodiments 45 to 49.

[0148] 53. A viral particle according to embodiment 52, further comprising SFV capsid and envelope proteins.

[0149] 54. A pharmaceutical composition comprising a nanobody as defined in any one of embodiments 1 to 6, or 21 to 26, or a nanobody conjugate as defined in any one of embodiments 7 to 20, or 27 to 40, or an expression vector as defined in any one of embodiments 45 to 49, a host cell as defined in any one of embodiments 50 to 51, or a viral particle as defined in any one of embodiments 52 to 53, together with a pharmaceutically acceptable excipient and / or carrier.

[0150] 55. A nanobody as defined in any one of embodiments 1 to 6, or 21 to 26, or a nanobody conjugate as defined in any one of embodiments 7 to 20, or 27 to 40, or an expression vector as defined in any one of embodiments 45 to 49, or a host cell as defined in any one of embodiments 50 to 51, or a viral particle as defined in any one of embodiments 52 to 53, or a pharmaceutical composition as defined in embodiment 54, for use in therapy.

[0151] 56. A nanobody as defined in any one of embodiments 1 to 6, or 21 to 26, or a nanobody conjugate as defined in any one of embodiments 7 to 20, or 27 to 40, or an expression vector as defined in any one of embodiments 45 to 49, or a host cell as defined in any one of embodiments 50 to 51, or a viral particle as defined in any one of embodiments 52 to 53, or a pharmaceutical composition as defined in embodiment 54, for the prevention and / or treatment of cancer.

[0152] Throughout the specification and claims, the word "comprise" and variations of this word are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the word "comprise" encompasses the term "consisting of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the specification or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to limit the present invention. Furthermore, the present invention covers all possible combinations of the specific preferred embodiments described herein. [Example]

[0153] Example 1. Local delivery of nanobodies targeting the PD-1 / PD-L1 axis by self-amplifying RNA viral vectors induces potent anti-tumor responses. method Cell lines and animals BHK-21 cells (ATCC-CCL10) were cultured in GMEM-BHK21 (Thermo Fisher, Waltham, MA) (complete GMEM) supplemented with 5% fetal bovine serum (FBS), 10% tryptose phosphate broth, 2 mM glutamine, 20 mM HEPES, and antibiotics (100 μg / mL streptomycin and 100 U / mL penicillin). HEK-293 cells (ATCC-CRL-3216) were cultured in DMEM (Gibco, BRL, UK) supplemented with 10% FBS, 2 mM glutamine, and antibiotics. MC38 cells, a gift from Dr. Karl E. Hellstrom (University of Washington, Seattle, WA), were cultured in RPMI-1640 medium (Lonza, Switzerland) supplemented with 10% FBS, 2 mM glutamine, 20 mM HEPES, antibiotics, and 50 μM 2-mercaptoethanol. B16-OVA cells were kindly provided by Dr. Lieping Chen and cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine, 20 mM HEPES, antibiotics, and 400 μg / mL Geneticin. Four- or 6-week-old female C57BL / 6 mice were purchased from Envigo (Barcelona, ​​Spain). Animal studies were approved by the Universidad de Navarra Ethics Committee for Animal Experiments under Spanish regulations (study number 078-19).

[0154] For the immunization and construction of the single-domain antibody library, one adult female llama (Lama glama) from the Montevideo Municipal Zoo (Uruguay) was used. The protocol was approved by the Parque Lecocq Ethics Committee, and handling of the llama was performed by a veterinarian.

[0155] In vitro PD-1 / PD-L1 inhibition assay The ability of nanobodies (Nbs) to inhibit the binding of PD-1 to PD-L1 was assessed by competitive ELISA. For human molecules, plates were coated with 0.5 μg / mL hPD-1-Fc (R&D, Minneapolis, MN) in PBS overnight at 4°C. After blocking with PBS-0.5% BSA, biotinylated hPD-L1 fused to human IgG Fc (BPS Bioscience, San Diego, CA) was added at 0.25 μg / mL along with various concentrations of anti-PD-1 Nbs or anti-PD-L1 Nbs (or mAbs) diluted in PBS-0.2% BSA. Detection of hPD-L1 bound to hPD-1 was performed using streptavidin conjugated to peroxidase. A similar protocol was followed for mouse molecules. In this case, plates were coated with mPD-L1-Fc (BioLegend, San Diego, CA) at 1 μg / mL in PBS, and biotinylated mPD-1-Fc (BPS Bioscience) was used at 0.25 μg / mL. These assays included commercially available antibodies as controls for PD-1 / PD-L1 blockade: anti-mouse PD-1 (clone RMPI-14, BioXCell, Lebanon, NH), anti-mouse PD-L1 (clone 10F.9G2, BioXCell), nivolumab (Bristol Myers Squibb, New York, NY), and atezolizumab (Roche, Basel, Switzerland). Signals in wells incubated without Nb or antibody were considered 100% PD-1 / PD-L1 binding.

[0156] Generation of SFV vectors encoding Nb The sequences of Nb11 (anti-PD-1) and Nb6p (anti-PD-L1) Nb were cloned into the SFVb12A plasmid using the ApaI restriction site [Rodriguez-Madoz JR, et al., Mol Ther. 2005] to generate the SFV-Nb11 and SFV-Nb6p vectors, respectively. These vectors contained a hemagglutinin (HA) tag at the carboxy terminus of each Nb sequence to enable protein detection. Nb sequences fused to the mouse Fc domain (including the hinge region) were synthesized using Genscript (Piscataway, NJ) and subcloned into the SFVb12A vector using the ApaI restriction site to generate the SFV-Nb11-Fc and SFV-Nb6p-Fc vectors, respectively. Nb11 was fused to mIgG1 Fc (Nb11-Fc), and Nb6p was fused to mIgG2a Fc (Nb6p-Fc). To generate SFV-aPD1, the sequence of an anti-PD-1 mouse monoclonal antibody (mAb) [US Patent No. 2010 / 0028330] was synthetically obtained from GenScript. Following the same strategy previously used to generate SFV-aPDL1, encoding a mAb against mouse PD-L1 with the same structure [Ballesteros-Briones MC, et al., Mol Ther. 2019], this synthetic gene, containing sequences corresponding to the heavy chain (IgG1 isotype) and light chain (lambda) of the aPD-1 mAb fused by the foot-and-mouth disease virus (FMDV) autoprotease 2A sequence preceded by a furin cleavage sequence, was subcloned into the ApaI site of SFVb12A. SFV-LacZ has been previously described [Quetglas JI, et al, Gene Ther. 2012].

[0157] For non-viral delivery experiments, the SFV-Nb11-Fc and SFV-LacZ replicons were subcloned into the pBK-T-SFV plasmid using the Apa I restriction site [Berglund P, et al. Nat Biotechnol. 1998].

[0158] Production of recombinant SFV viral particles SP6 RNA polymerase (Promega) and m 7 SFV RNA was transcribed in vitro from the SFV plasmid using a G(')ppp(5')G RNA Cap Structure Analog (New England Biolabs, Ipswich, MA). RNA synthesis by electroporation and delivery into BHK-21 cells were performed as previously described [Liljestrom P, et al. Curr Protoc Mol Biol. 2001]. To produce SFV virus particles, two helper RNAs (SFV-Helper-C-S219A and SFV-Helper-S2) were co-electroporated with the recombinant RNA, resulting in the expression of SFV capsid and envelope proteins in trans [Smerdou C, et al. J Virol. 1999]. Forty-eight hours after electroporation, the supernatant was collected, and virus particles were purified by ultracentrifugation as described [Ballesteros-Briones MC, et al. Mol Ther. 2019]. For viral particle titration, BHK-21 cells were infected with serial dilutions of SFV vectors. For SFV-Nb11 and SFV-Nb6p, indirect immunofluorescence was performed using a mouse anti-HA primary antibody (BioLegend) and a secondary anti-mouse IgG antibody conjugated to Alexa-488 (Invitrogen, Waltham, MA). For antibodies containing the Fc domain, direct immunofluorescence was performed using the same anti-mouse IgG antibody. For SFV-LacZ, infected cells were treated with X-Gal staining solution, and blue cells were counted as positive. Vector titers ranged from 2 to 6 × 10 for SFV vectors encoding Nb or antibody. 10 virus particles / mL, 0.5–2 × 10 for SFV-LacZ 11 The range was 0.01 to 0.1 viral particles / mL.

[0159] Expression of Nb from SFV vectors in vitro BHK-21 cells infected with SFV viral particles were used to evaluate the expression of Nb in vitro.6 BHK-21 cells were seeded in 6-well plates and, 24 hours later, infected with SFV viral particles at a multiplicity of infection (MOI) of 20. Viral particles were diluted in 300 μL of MEM supplemented with 2 mM glutamine and 0.2% BSA, and cells were infected at 37°C for 1 hour. The infection medium was then replaced with complete GMEM. To evaluate the expression of Nb11-Fc from the pBK-T-SFV plasmid, 5 × 10 cells were infected in 6-well plates using Lipofectamine-2000 (Thermo Fisher). 5 BHK-21 cells / well were transfected with 2 μg of plasmid / well.

[0160] Supernatants and cell extracts from infected or transfected cells were collected 24 hours later to assess antibody expression. Supernatants were centrifuged at 10,000 g for 5 minutes at 4°C to remove cell debris. Cells were washed three times with PBS and then incubated with cold lysis buffer (50 mM Tris-HCl pH 7.5, 1% NP-40, 150 mM NaCl, 2 mM EDTA, and Protease Inhibitor Cocktail, Roche) for 10 minutes at 4°C. Lysed cells were centrifuged at 6,000 g for 10 minutes at 4°C, and the supernatant (containing proteins from the lysed cells) was collected for analysis. Samples were stored at -80°C until use.

[0161] Antibody quantification by ELISA To quantify monomeric Nb in samples from in vitro and in vivo experiments, specific hPD-1 or mouse PD-L1 (mPD-L1) binding ELISAs were performed. 96-well ELISA plates were coated overnight at 4°C with hPD-1 fused to human IgG1 Fc (R&D) at 0.2 μg / mL or mPD-L1 fused to human IgG1 Fc (BioLegend) at 1 μg / mL in PBS. The following day, plates were blocked with 0.5% BSA in PBS at room temperature for 1 hour, washed with PBST, and incubated with samples diluted in PBST-0.2% BSA at room temperature for 2 hours. Detection was performed using an anti-HA antibody (BioLegend) followed by incubation with a peroxidase-conjugated polyclonal antibody against mouse IgG (Sigma, St. Louis, MO). Standard curves were included in all assays using the corresponding purified Nb produced in E. coli as previously described [Silva-Pilipich N, et al. Biomedicines. 2020].

[0162] To compare the expression of the Nb-Fc constructs from in vitro experiments with conventional mAbs, samples were analyzed by sandwich ELISA for the detection of total mouse IgG. ELISA plates were coated overnight at 4°C with polyclonal anti-mouse IgG (Abcam, UK) in PBS, blocked for 1 hour at room temperature with 0.5% BSA in PBS, and incubated with samples diluted in PBST-0.2% BSA for 2 hours at room temperature. Antibodies conjugated to peroxidase against mIgG1 or mIgG2a were used for detection (Abcam, UK). The assay was developed using tetramethylbenzidine (TMB) substrate and stopped with H2SO42N. Absorbance was read at 450 nm using a Fluostar Optima Reader. A standard curve was included in all assays using purified Nb11-Fc or Nb6p-Fc from the supernatant of BHK-21 cells electroporated with SFV-Nb11-Fc RNA and SFV-Nb6p-Fc RNA. Nb6p-Fc was purified using a Protein A Sepharose column and the AKTA purification system (Cytiva, Marlborough, MA) according to the manufacturer's instructions. Nb11-Fc was concentrated using a 50 kDa Amicon filter (Millipore, Burlington, MA). Nb quantification was performed by Coomassie blue staining using BSA as a standard.

[0163] Western blot analysis of antibodies For both Fc-fused Nbs and mAbs, samples from BHK-21 cells infected with SFV vectors were analyzed by Western blotting under reducing (with dithiothreitol, DTT) or non-reducing (without DTT) conditions on 10% or 8% polyacrylamide gels, respectively. For monomeric Nbs, 15% polyacrylamide gels were used due to their small size. Detection of monomeric Nbs or Fc-fused Nbs was performed using the same antibodies described for the ELISA assay, diluted in TBS-0.05% Tween 20-5% nonfat milk. The membranes were developed using Lumigen ECL Ultra substrate (Beckman Coulter, Brea, CA), and images were acquired using a ChemiDoc Imaging System (BioRad, Hercules, CA).

[0164] Tumor induction and treatment C57BL / 6J mice were injected with 5 × 10 PBS diluted in saline into the right flank. 5 Tumor cells were injected subcutaneously (sc). Seven to 10 days after tumor inoculation, SFV vectors diluted in saline were administered intratumorally (it) in a total volume of 50 μL. All experiments, except for the bilateral tumor model, included one dose of 3 × 10 cells. 8 For bilateral tumor experiments, 5 × 10 virus particles were administered to the right and left flanks, respectively. 5 and 3 x 10 5 MC38 cells were inoculated. Two doses of SFV-Nb11-Fc (3 × 10) were administered 5 days apart. 8 The largest tumors were treated with 1000 viral particles / dose.

[0165] Evaluate the efficacy of treatment by measuring two perpendicular tumor diameters every 2-3 days, using the formula: volume = (length × width) 2 Tumor volume was calculated using the formula: ) / 2. 3 Mice were humanely sacrificed when they reached a mass of 1000 mg / kg or when tumor ulceration or obvious discomfort was observed.

[0166] For rechallenge experiments in the MC38 model, mice that had rejected tumors were inoculated with 5 × 10 cells into the left flank 3 months after the initial tumor inoculation. 5 MC38 cells were injected sc. Naive mice were included as controls and tumor development was assessed for 2 months.

[0167] Expression of Nb from SFV vectors in vivo To evaluate in vivo Nb expression from the SFV vector, mice were sacrificed 1 and 5 days after VP administration, and tumor, blood, and urine samples were collected and analyzed by ELISA. Blood samples were obtained by retroorbital sinus bleeding, incubated at room temperature for 30 minutes, and then centrifuged at 3,000 rpm for 10 minutes. The supernatant (i.e., serum sample) was collected for analysis. Urine samples were centrifuged at 10,000 rpm for 2 minutes, and the supernatant was collected for analysis. Tumors were weighed and homogenized in 3 volumes of PBS-0.05% Tween 20 supplemented with Protease Inhibitor Cocktail using a homogenizer. Samples were incubated with shaking at 4°C for 1 hour, centrifuged at 10,000 rpm for 15 minutes, and the supernatant was collected for analysis. Total protein quantification for normalization was performed using a BCA kit (Pierce, Appleton, WI) according to the manufacturer's instructions.

[0168] Flow cytometry analysis The immune response after treatment with SFV vectors encoding Nb11-Fc or Nb6p-Fc was characterized in the MC38 tumor model. Five days after treatment with various SFV vectors, draining lymph nodes and tumors were collected from each mouse. Lymph nodes were homogenized in PBS using a 70 μm filter. Excised tumors were digested with 400 U / mL collagenase D and 50 μg / mL DNase-I (Roche) at 37°C for 20 minutes and then homogenized using a 70 μm cell strainer. Samples were washed with PBS, centrifuged, and the pellets were resuspended in PBS for staining. For functional analysis, cells were incubated with Zombie NIR Fixable viability dye (Biolegend). They were then stained with anti-CD16 / 32 antibody (clone 2.4G2, BD Biosciences). Blocking was performed with Pharmingen (Pharmingen) and the following antibodies were used: CD45.2-APCA750 (clone 104), CD3-BV605 (clone 17A2), CD8a-BV510 (clone 53-6.7), CD4-APCAF700 (clone RM4-5), ICOS-PerCP-Cy5.5 (clone 7E.17G9), CD137-APC (clone 17B5), TIM3-BV785 (clone RMT3-23), LAG3-B Cells were stained with fluorochrome-conjugated mAbs against V650 (clone C9B7W), CD11b-PeCy7 (clone M1 / 70), CD11c-APC (clone N418), Ly6C-BV510 (clone HK1.4), F4 / 80-BV421 (clone BM8), PD-L1-BV785 (clone 10F.9G2) (all Biolegend), PD-1-FITC (clone RMP1-30, Invitrogen), Ly6G-PE (clone 1A8, BD Biosciences), and NKp46-BV650 (clone 9-E2, BD Biosciences). For intracellular staining, cells were fixed and permeabilized with BD Fixation / Perm buffer (BD Biosciences) and then stained with anti-granzyme B-BV421 mAb (clone GB11, Biolegend) and anti-KI67-PE-Cy7 mAb (clone 16A8, BD).+ To identify T lymphocytes, cells were stained with PE-labeled H-2Kb MuLV p15E Tetramer-KSPWFTTL (MBL International, Woburn, MA). Samples were acquired using a CytoFLEX cytometer (Beckman Coulter), and data were analyzed using FlowJo software (TreeStar, Ashland, OR).

[0169] RNA sequencing analysis 3 x 10 of SFV-Nb11-Fc or SFV-LacZ 8 C57BL / 6J mice bearing MC38 sc tumors were treated with a single it dose of viral particles / tumor. Control, untreated mice received the same volume of saline. Five days after treatment, mice were sacrificed, and tumor samples were collected and homogenized in 2 mL of TRIzol reagent (Sigma-Aldrich). RNA was isolated according to the manufacturer's instructions and further purified using an RNeasy Mini Kit (Qiagen). The quality of the purified RNA was assessed using the High Sensitivity RNA ScreenTape system (Agilent, Santa Clara, CA). Five samples with an RNA integrity number (RIN) >7 per group were selected, and RNA samples were prepared using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) according to the manufacturer's instructions. Briefly, polyA RNA from the samples was enriched using polyT oligo-conjugated magnetic beads, fragmented, and reverse transcribed using random primers. Double-stranded cDNA samples were end-repaired, adenylated, and ligated to TruSeq adapters containing indexes for multiplexing. After PCR amplification of fragments, libraries were sequenced using an Illumina NextSeq2000 (Illumina).

[0170] The workflow for RNAseq data analysis was as follows: 1) sample quality control using FastQC software (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), 2) read alignment to the mouse genome (mm10) using STAR [Dobin A, et al. Bioinformatics. 2013;29:15-21], 3) gene expression quantification using featureCounts [Liao Y, et al. Bioinformatics. 2014], 4) the gene annotation reference used was Gencode M25 [Harrow J, et al. Genome Res. 2012], and 5) differential expression statistical analysis using R / Bioconductor [Gentleman RC, et al. Genome Biol. 2004].

[0171] Gene expression data were normalized using edgeR [Robinson MD, et al. Bioinformatics. 2010]. Genes with read counts less than 6 in more than 50% of samples were not considered in this experiment. LIMMA [Ritchie ME, et al. Nucleic Acids Res. 2015] was used to identify genes with significant differential expression between experimental conditions using a cutoff of p<0.05. Further functional analysis was performed using Gene Set Enrichment Analysis (GSEA) with the MsigDB C7 collection of gene sets [Subramanian A, et al. Proc Natl Acad Sci. 2005]. In this analysis, p<0.01 and a false discovery rate (FDR)<0.05 were considered statistically significant.

[0172] Non-viral delivery of SFV by electroporation Six-week-old female C57BL / 6J mice were injected with 5 × 10 5MC38 cells were injected sc. Ten days later, intratumoral delivery of pBK-T-SFV plasmids carrying SFV replicons containing the Nb11-Fc or LacZ gene was performed by electroporation as previously described (Silva-Pilipich N, et al., Molecular Therapy Nucleic Acids, 2022) with some minor modifications. Briefly, 2 hours before treatment, hyaluronidase type IV (Sigma) was injected it (30 units / tumor). 20 μg of pBK-T-SFV endotoxin-free plasmid was used per dose, diluted in 25 μL of PBS. Mice were anesthetized and injected it with the plasmids. Immediately thereafter, local electroporation was performed using an ECM 830 electroporation system (BTX, Holliston, MA) with the following conditions: eight pulses of 1200 V / cm, each 0.1 ms in duration, spaced 5 ms apart. For the first treatment, a simple surgical procedure was performed to expose the tumor, and this procedure was repeated three times every three days.

[0173] statistical analysis Data are expressed as mean ± SD or mean ± SEM, as specified in each figure legend. Statistical analysis was performed using Prism software (GraphPad Software, San Diego, CA). One-way ANOVA and Tukey's multiple comparison test were used to compare multiple experimental groups. A two-tailed Student's t-test was applied to compare two experimental groups. For time series analysis, data were compared using an additional sum-of-squares F-test and fitted to a second-order polynomial equation. The survival rate of tumor-bearing mice is depicted by Kaplan-Meier plots and analyzed by the log-rank test. A p-value <0.05 was considered statistically significant.

[0174] result Characterization of PD-1 and PD-L1 specific Nbs To evaluate their ability to inhibit PD-L1 binding to PD-1, we performed inhibition ELISAs using commercially available PD-1 and PD-L1 ectodomains fused to a human IgG1 Fc domain. The Nb11 and Nb6p nanobodies were able to inhibit human PD-1 binding to PD-L1 (Figure 8A, B). In a similar inhibition ELISA, we also evaluated the ability of these nanobodies to inhibit the binding of their mouse orthologs and found that these two nanobodies were able to inhibit PD-1 / PD-L1 binding by more than 50% at 300 nM (Figure 8C). Nb6p, in fact, demonstrated significantly superior inhibitory activity, comparable to that of an anti-mouse PD-L1 antibody that previously demonstrated potent antitumor activity (Figure 8D) [Ballesteros-Briones MC, et al., Mol Ther. 2019].

[0175] Evaluation of SFV vectors encoding monomeric Nbs against PD-1 and PD-L1 In this study, we developed SFV vectors expressing both the Nb11 and Nb6p Nbs to test their antitumor potential through localized delivery to tumors. To this end, the Nb11 and Nb6p sequences were cloned into SFV to generate the SFV-Nb11 and SFV-Nb6p vectors, respectively. In both cases, the Nb sequences were designed to have a signal peptide at the amino terminus and a hemagglutinin tag at the carboxyl terminus for detection (Figure 1A). Nb expression in BHK-21 cells infected with SFV-Nb11 and SFV-Nb6p viral particles was assessed at 24 h postinfection. The SFV-LacZ vector was used as a control. Immunofluorescence (IF) analysis of infected cells demonstrated coexpression of both Nb and SFV replicase (results not shown). Western blot analysis showed that both Nbs were expressed at the expected size (approximately 15 kDa) in cell extracts but displayed a higher molecular weight (MW) in the supernatant (Fig. 1B). This is consistent with previous observations for Nb11 expressed from an AAV vector plasmid, where we demonstrated that this change was due to glycosylation and did not affect antigen binding

[18] . Nb quantification was performed by specific ELISA for PD-1 or PD-L1, and in both cases the majority of Nb (approximately 85%) was present in the supernatant, confirming highly efficient secretion. Expression levels were measured in 10 infected cells. 6 Approximately 20 μg and 10 μg of Nb11 and Nb6p were secreted per cell, respectively (Fig. 1C).

[0176] We then evaluated the antitumor activity of the SFV-Nb11 and SFV-Nb6p vectors in a mouse colon adenocarcinoma model using subcutaneously implanted MC38 cells. We used an SFV vector encoding a full-length anti-PD-L1 mAb (SFV-aPDL1) at a dose of 3 × 10 , a dose that previously demonstrated significant antitumor activity in this model. 8Tumors were treated intratumorally with SFV-Nb11 and SFV-Nb6p virus particles [Ballesteros-Briones MC, et al., Mol Ther. 2019]. Antitumor effects were achieved in vivo using the SFV-Nb11 and SFV-Nb6p vectors, and some delay in tumor growth rate was observed (Figure 1D, E).

[0177] Generation of SFV vectors expressing optimized Nb We modified SFV vectors expressing Nb11 and Nb6p by fusing each Nb sequence to the IgG Fc domain to promote their dimerization. To this end, Nb11 was fused to the mouse IgG1 Fc domain (generating SFV-Nb11-Fc). This isotype, lacking its secondary function as PD-1, is primarily expressed on T cells (Figure 2A). Nb6p was fused to the mouse IgG2a Fc domain to generate SFV-Nb6p-Fc (Figure 2A).

[0178] We first analyzed the expression of Nb-Fc fusion molecules in BHK-21 cells infected with the SFV-Nb11-Fc and SFV-Nb6p-Fc vectors. In this experiment, SFV vectors expressing conventional mAbs against mouse PD-L1 and PD-1 (SFV-aPD1 and SFV-aPDL1, respectively [Ballesteros-Briones MC, et al., Mol Ther. 2019]) were used as controls. As previously observed, infected cells demonstrated coexpression of both Nb-Fc and SFV replicase by IF (results not shown). Quantification of recombinant antibodies from infected cells by ELISA revealed significantly higher expression of Nb-Fc molecules compared to mAbs (Figure 2B). Although the expression levels of dimeric Nbs were two- to four-fold lower than those observed for the monomeric forms (Figure 1C), they were also efficiently secreted (Figure 2B). Both Nb11-Fc and Nb6p-Fc were able to dimerize when analyzed by Western blot using non-reducing conditions (Fig. 2C, bottom panel). Under reducing conditions, both Nb-Fc molecules exhibited the predicted MW (approximately 40 kDa, Fig. 2C, top panel).

[0179] Fusion of Nbs to the Fc domain may affect their antigen-binding properties, as dimerization can increase their binding activity. To test whether this is the case, we generated inhibition curves of PD-1 / PD-L1 binding using monomeric Nbs and Fc-fused versions prepurified from SFV-transfected cells as described in the methods. Interestingly, fusion to the Fc domain improved the inhibitory potency of Nbs for mouse and human molecules, with approximately 40-fold and 7-fold reductions in IC50 for Nb11-Fc and Nb6p-Fc, respectively (Figure 3). We also included commercially available mAbs in these assays and observed that Nb6p-Fc had a similar IC50 compared to atezolizumab and clone 10F-9G2, an anti-mouse PD-L1 mAb commonly used in preclinical studies [Grasselly C, et al., Front Immunol. 2018]. For Nb11-Fc, a 10-fold and 700-fold reduction in IC50 was observed compared to nivolumab and clone RMPI-14, respectively, an anti-mouse PD-1 mAb that showed potent antitumor activity in preclinical studies [Grasselly C, et al, Front Immunol. 2018; Ngiow SF, et al Cancer Res 2015].

[0180] Antitumor activity of SFV vectors expressing Nb-Fc Because Nb-Fc fusions resulted in lower IC50 values ​​for PD-1 / PD-L1 inhibition, we reasoned that these molecules might have superior performance in vivo compared with monomeric Nbs. For comparison, we evaluated SFV vectors encoding Nb11-Fc and Nb6p-Fc, including vectors encoding monomeric Nbs (SFV-Nb11 and SFV-Nb6p) and conventional mAbs (SFV-aPD1 and SFV-aPDL1), in an MC38 subcutaneous tumor model. SFV-Nb11-Fc and SFV-Nb6p-Fc demonstrated highly potent antitumor effects, delaying tumor growth and significantly improving survival compared with controls (saline and SFV-LacZ) (Figure 4, left panel). Treatment with SFV-Nb11-Fc and SFV-Nb6p-Fc resulted in long-term survival rates of 53% and 60%, respectively, in contrast to the 20% and 28% obtained with SFV-aPD1 and SFV-aPDL1, respectively (Figure 4, center panel). As previously observed, monomeric Nbs exhibited modest antitumor effects, similar to or lower than those obtained with conventional antibodies. Treated animals that achieved complete remission remained tumor-free even after rechallenge with MC38 cells (Figure 4, right panel), suggesting that these treatments were able to generate efficient memory immune responses.

[0181] The efficacy of SFV-Nb11-Fc and SFV-Nb6p-Fc was also verified in a melanoma tumor model (B16-OVA), and significant delay in tumor growth and improved survival rate were observed compared to the saline and SFV-LacZ groups (Figure 9).

[0182] One of the main challenges of local immunotherapy is to induce a systemic antitumor effect. To evaluate whether SFV expressing dimeric Nb can produce an abscopal effect in untreated tumors, we used a bilateral subcutaneous MC38 tumor model. Two intratumoral doses of SFV-Nb11-Fc vector (3 × 10) were injected into one of the nodules of the mice at 5-day intervals. 8The vector resulted in a significant reduction in the size of treated tumors, but had a relatively modest effect in controlling the growth of untreated nodules. Untreated tumors in mice administered with SFV-Nb11-Fc showed a slight delay in growth from day 9 after the first administration compared with control mice administered only saline, but the difference was not significant (Fig. 10A). Nevertheless, treating only one tumor nodule with SFV-Nb11-Fc was sufficient to significantly increase the survival rate of mice compared with controls (Fig. 10B), suggesting that this type of treatment can induce an abscopal effect.

[0183] In vivo expression of Nb from SFV vectors Next, we evaluated the levels and persistence of various Nbs expressed from SFV administered locally to MC38 tumors using the same doses as in the treatment experiments. Expression of only monomeric and Fc-fused Nbs was observed in tumor tissue on day 1 after treatment, whereas Nb levels were undetectable in any mouse by day 5 (Figure 5A). This is consistent with previous observations using SFV vectors. In most cases, Nbs were undetectable in the blood, but significant leakage into the systemic circulation was observed with Nb11-Fc on day 1 after injection, with levels of approximately 180 ng / mL (Figure 5B). This could be due to higher intratumoral expression of this Nb than Nb6p-Fc, or the fact that anti-PD-L1 Nbs may be more efficiently retained in the tumor microenvironment than anti-PD-1 Nbs due to high PD-L1 expression in MC38 tumors

[10] . In the case of monomeric Nbs, they could also be detected in the urine, with a slight increase observed on day 1, suggesting that these small molecules could be eliminated from the circulation through renal clearance (Figure 5C).

[0184] Antitumor immune responses elicited by SFV encoding dimeric Nb To evaluate the antitumor immune response induced by SFV vectors encoding Nb-Fc nanobody conjugates, MC38 tumor-bearing mice were treated with SFV-Nb11-Fc and SFV-Nb6p-Fc vectors, as well as SFV-LacZ and saline as controls, and sacrificed 5 days later. Tumor and draining lymph node (dLN) samples were processed and analyzed by flow cytometry using various markers. In the case of SFV-Nb11-Fc, mice had very small tumors at the time of sacrifice and could not be included in this analysis (Figure 6A). However, we analyzed changes in the dLN for both Nb groups.

[0185] In tumors treated with SFV-Nb6p-Fc, total CD8 + A significant increase in T cells was observed, expressing high levels of the activation marker ICOS and showing a tendency to express high levels of CD137 and granzyme b (Figure 6B). Total MuLV tetramer-specific CD8+ T cells in the SFV-Nb6p-Fc group were significantly higher than those in the control group. + No relevant changes were observed in T cells, although these cells showed a significant increase in the exhaustion marker TIM-3, suggesting a more potent activation state of this population (Figure 6C). + T cells and MuLV-specific CD8 + No significant changes in PD-1 expression were observed in T cells (Fig. 6D). + No overall changes in T cell populations were observed (data not shown). A higher percentage of NK cells (NKp46) was observed in tumors treated with SFV-Nb6p-Fc than in the saline and SFV-LacZ groups. + cells) and CD11b + However, macrophages (F4 / 80 + CD11b + No changes were observed in granulocytes (Ly6G + CD11b + cells) was significantly reduced (Figure 6E).

[0186] In dLN, total CD8 +Although no overall changes were observed for T cells, this population displayed significantly lower levels of PD-1 in mice treated with both SFV-Nb-Fc vectors, which may indicate a less exhausted phenotype in this location (Fig. 11A). However, these cells displayed lower levels of ICOS. In contrast to what was observed in the tumor, CD11b expression in the dLN was significantly higher. + The cells showed a tendency to decrease in response to both Nb-Fc treatments, with no change in PD-L1 (Fig. 11B). + CD11b + A significant increase in SFV-Nb11-Fc cells was observed, and a similar trend was observed with SFV-Nb11-Fc (Figure 11B). Interestingly, these cells expressed low levels of PD-L1 in both Nb-Fc groups.

[0187] Tumor samples from animals treated with SFV-Nb11-Fc, SFV-LacZ, and saline were used for bulk RNA sequencing analysis to gain further insight into altered immunological pathways. Several immune-related genes were found to be differentially expressed in tumors treated with SFV-Nb11-Fc compared to tumors injected with saline. The SFV-LacZ vector was also able to induce significant transcriptome changes in three of the five tumors analyzed, underpinning the importance of SFV vectors in immune modulation. However, the changes were relatively uniform and therefore more pronounced in tumors treated with SFV-Nb11-Fc. As expected, several viral stress-inducible genes (Sting1, Batf2, Nod1, Tirap, Ikbke, Clec4d, Clec4e, and Irf1) were found to be upregulated in SFV-treated tumors. In addition, SFV-Nb11-Fc treatment resulted in the upregulation of various immune stimulatory genes, including cytokines (Il2g, Il2ra, Il15ra, Il18rap, Ifng, Tgfa), chemokines (Cxcl9, Cxcl10, Cxcl11), and IFN-I response genes (Stat1). The upregulation of molecules involved in cell adhesion and motility (Icam1, Itgal, Selp, etc.) suggests an increased recruitment of immune cell populations to the tumor site. NK cells and cytotoxic CD8 + Several genes associated with enhanced T cell activity (Ncr1, Nkg7, Prf1, Gzma, Gzmb, Gzmk) and genes expressed upon TCR stimulation (Nfatc2, Lat, Cd6, Cd5) were also upregulated, suggesting increased infiltration and activation of these effector cells. Increased expression of genes associated with apoptosis (Cd40, Cd40lg, Fasl, Ripk1) was also observed. Finally, SFV-Nb11-Fc treatment resulted in downregulation of genes associated with angiogenesis (Vegfa and Jmjd8) and metastasis-promoting factors (Cxcr4, Mmp11, Macc1). The latter is a known marker of poor prognosis in various cancer types, including colorectal cancer.

[0188] Using the gene set enrichment analysis (GSEA) tool [Subramanian A, et al., Proc Natl Acad Sci 2005] and the C7 database, we further analyzed the transcriptome differences between tumors treated with SFV-Nb11-Fc and SFV-LacZ to compare changes in immunological signatures. Significant differences were found between both treatment groups, and many of the altered gene sets were related to CD8 expression in various cells. + It was associated with T cell activation, NK function and IFNγ signaling.

[0189] Non-viral delivery of SFV vectors encoding anti-PD-1 dimeric Nbs An alternative to using SFV viral particles would be to deliver SFV vectors to tumors using non-viral methods. To test whether this delivery system could be used with SFV vectors expressing dimeric Nbs, we generated a plasmid (pBK-SFV-Nb11-Fc) carrying the SFV-Nb11-Fc replicon under the transcriptional control of the CMV promoter (Figure 7A). Expression of Nb11-Fc from this plasmid was confirmed in vitro by Western blot analysis of transfected BHK-21 cells (Figure 7B). As diagrammed in Figure 7C, mice bearing MC38 subcutaneous tumors were administered three doses of pBK-SFV-Nb11-Fc plasmid (20 μg / dose) every three days, followed by local electroporation as previously described (Silva-Pilipich N, et al., Molecular Therapy Nucleic Acids, 2022). This treatment protocol resulted in a significant reduction in tumor growth compared to untreated controls or mice administered the SFV-LacZ plasmid by electroporation (Figure 7D). A significant increase in survival was also achieved in animals administered pBK-SFV-Nb11-Fc compared to untreated mice (Figure 7E). Similar to mice treated with viral vectors, this strategy resulted in 100% protection after MC38 tumor rechallenge, suggesting that this non-viral delivery system can also promote long-lasting anti-tumor immune responses.

[0190] Consideration Antibodies capable of blocking the PD-1 / PD-L1 axis, such as nivolumab, pembrolizumab, and atezolizumab, have shown remarkable therapeutic efficacy in patients with various tumor types [Xiang Z, et al., Front Pharmacol. 2022]. However, frequent adverse effects observed in patients treated with checkpoint inhibitors and the lack of response in some tumor types necessitate the improvement of these treatments [Sun G, et al., Int J Oncol. 2022]. In this study, we addressed these issues by developing a strategy based on local intratumoral delivery of nivolumab using a self-amplifying RNA vector that can induce type I IFN responses and apoptosis in tumor cells, favoring a response in tumors that typically do not respond well to immunotherapy.

[0191] We developed novel Nbs against PD-L1 (Nb6p) and PD-1 (Nb11) that can block the interaction between PD-1 and PD-L1 in both mouse and human molecules. The blocking efficacy of Nb6p and Nb11 was improved by homodimerization with the IgG Fc domain, resulting in a significant reduction in IC50 in PD-1 / PD-L1 binding assays (approximately 8-fold and 40-fold reductions for Nb6p and Nb11, respectively, in both mouse and human interactions). Notably, Nb11-Fc exhibited an IC50 8.6-fold lower than that of nivolumab, while Nb6p-Fc had an IC50 very similar to that of atezolizumab. These results indicate that these newly described dimeric Nbs may have clinical potential as recombinant proteins. A similar anti-PD-L1 camelid-derived Nb fused to human IgG1 Fc, called emvafolimab, has recently been approved in China for the treatment of various solid tumors and is being tested in the United States for soft tissue sarcomas and biliary tract cancers [Markham A. Drugs 2022]. Interestingly, emvafolimab can be administered subcutaneously and has a high safety profile. Embafolimab exhibits an IC50 of 5.25 nM for blocking PD-L1 PD-1 interactions [Markham A., Drugs 2022], approximately 10-fold higher than that observed with Nb6p-Fc.

[0192] Although dimeric Nbs are larger than their corresponding monomeric forms, their size is approximately half that of conventional antibodies, which may confer superior tumor penetration. An additional advantage of dimeric Nbs is the possibility to modulate their function using different Fc domains.

[0193] The improved blocking activity exhibited by dimeric Nbs in vitro was recapitulated in vivo when expressed from intratumorally delivered SFV vectors. Indeed, SFV vectors expressing monomeric Nbs have already demonstrated antitumor activity in MC38 colon adenocarcinoma tumors. SFV vectors expressing either Nb11-Fc or Nb6p-Fc had potent antitumor effects in both colon and melanoma mouse tumors, resulting in over 50% complete regression in the first model using both vectors. This effect was superior to that obtained using SFV vectors expressing conventional anti-PD-L1 and anti-PD-1 mAbs, which also showed significant antitumor efficacy, as previously described [Ballesteros-Briones MC, et al., Mol Ther. 2019]. One possible reason for the superior performance of SFV vectors carrying dimeric Nbs may be the much higher expression of these molecules compared to mAbs, as observed in vitro in infected cells (Figure 2C). In vivo Nb expression was highly transient with both vectors and was primarily detected 1 day after treatment. Expression appeared to be tumor-restricted, but relatively high levels were transiently observed in serum with Nb11-Fc, suggesting that some dimeric Nb may leak from tumors. However, this effect was not observed with Nb6p-Fc, likely due to the high expression of PD-L1 in MC38 tumors. Low levels of monomeric Nb were observed in urine 1 day after vector administration. In our study, Nb was expressed locally, but extremely low plasma levels of monomeric Nb were detected, suggesting that the fraction leaking from tumors would be rapidly eliminated via urine, as was indeed the case. However, extremely low levels of dimeric Nb were detected in urine, confirming that the Fc domain may increase their half-life in serum.

[0194] A notable feature of this SFV-based therapy was that a single, extremely short-term administration of a vector locally expressing a dimerized Nb was able to promote a potent and long-lasting antitumor response. Several immune-stimulatory genes were significantly upregulated in tumors treated with SFV-Nb11-Fc compared with the saline control group. Expression of a dimerized Nb capable of blocking the PD-1 / PD-L1 axis appeared to be important for eliciting a curative immune response.

[0195] SFV viral particles can be produced very efficiently, and a recent clinical trial in cervical cancer patients using an SFV vector expressing human papillomavirus (HPV) E6 and E7 proteins demonstrated that they may have a high degree of safety in humans [Komdeur FL, et al., Mol Ther. 2021]. However, clinical implementation of viral particles may be more challenging compared to the use of nonviral vectors, which are less expensive to produce at GMP levels and may have a higher safety profile. The SFV system has the advantage of being able to be used in a nonviral manner, either as an RNA or DNA / RNA layered plasmid vector. We chose this latter option to evaluate one of two SFV vectors expressing dimerized Nb (pBK-SFV-Nb11-Fc). Interestingly, local delivery of this plasmid to MC38 tumors by electroporation resulted in antitumor effects comparable to those obtained with viral particles. An additional advantage of the DNA system is that it does not induce neutralizing antibodies against SFV and allows for multiple administrations.

[0196] Example 2. Immunoconjugates containing anti-PD-1 nanobodies and anti-PD-L1 nanobodies fused to interleukin-12 Construction of SFV vectors expressing fusion proteins of IL-12 with nanobodies Nb6p and Nb11 To achieve even greater antitumor efficacy, we constructed SFV vectors expressing IL-12 fused to the Nb11 nanobody (Nb) and the Nb6p nanobody (Nb) according to the scheme shown in Figure 12. To do this, we used a single-chain version of IL-12 (scIL12) [GJ Lieschke, et al., Nat. Biotechnol. 15 (1997)] with each nanobody fused at either the amino or carboxy terminus, generating four SFV vectors expressing the following fusion proteins: scIL12-Nb11, scIL12-Nb6p, Nb11-scIL12, and Nb6p-scIL12 (Figure 12A). A different type of construct was based on a two-chain version of IL-12 (dcIL12), in which each subunit of IL-12 (p35 or p40) was fused to an Nb [Rodriguez-Madoz JR, et al., Mol Ther. 2005] (in this case, generating IL-12 with two copies of an Nb). In this case, three SFV vectors were generated expressing the following fusion proteins: dcIL12-Nb11, dcIL12-Nb6p, and dcIL12-bis (which contains p35 fused to Nb6p and p40 fused to Nb11) (Figure 12B).

[0197] ICK activity in vitro To test the activity of ICK, viral particles were generated for each of the aforementioned SFV vectors. BHK cells were then infected with viral particles from each SFV vector at a multiplicity of infection (MOI) of 10, using cells infected with the same amount of SFV-LacZ [Quetglas JI, et al., Gene Ther. 2012] as a negative control. In this assay, SFV vectors expressing scIL12 and dcIL12 were also used as positive controls [Rodriguez-Madoz JR, et al., Mol Ther. 2005]. After 24 hours, supernatants and cell extracts were collected, and the amount of IL-12 was quantified using a commercially available ELISA kit (BD Biosciences). As shown in Figure 13A, SFV-scIL12-Nb11 and SFV-Nb11-scIL12 expressed IL-12 at levels similar to SFV-scIL12 (approximately 50 μg / 10 ). 6 SFV-scIL12-Nb6p and SFV-Nb6p-scIL12 expressed IL-12 at a lower level (approximately 30 μg / 10 cells). 6 The expression level was high (approximately 40 μg / 10 cells). 6 In the case of vectors expressing dcIL12-Nb fusions, all of them were significantly higher than the control SFV-dcIL12 vector (approximately 20 μg / 10 cells), except for SFV-dcIL12-Nb11, which showed a similar effect to the control SFV-dcIL12 vector (approximately 20 μg / 10 cells). 6 The expression levels were similar to those of the control cells.

[0198] To test the activity of IL-12 fusion proteins, splenocytes were extracted and isolated from healthy mice. Animals were sacrificed, and spleens were collected in 10 ml of PBS or unsupplemented RPMI 1640 medium. Tissue disruption was performed using a cell strainer. The final pellet was resuspended in 1 ml of RPMI medium supplemented with 10% FBS and antibiotics. 2 x 10 IL-12 fusion proteins diluted in 1 ml of RPMI medium were added per well of an M24 plate. 6Splenocytes were seeded onto the SFV-ICK virus particles. To stimulate IFNγ production, supernatant from cells infected with SFV-ICK virus particles was incubated with mouse splenocytes. Incubation was carried out at 37°C for 48 hours. Supernatant from cells infected with SFV-IL-12 PV was used as a positive control, and supernatant from untransfected BHK cells or complete RPMI 1640 medium was used as a negative control. After incubation, samples were centrifuged at 6000 rpm for 5 minutes, and the supernatants were collected and quantified by a commercially available IFNγ-specific ELISA (Mouse IFNγ ELISA Set, BD Biosciences). As seen in Figure 13B, all ICKs induced similar levels of IFNγ, indicating that they were all equally active.

[0199] ICK activity in vivo C57BL / 6J mice were injected with 5 × 10 PBS diluted in saline into the right flank. 5 10 tumor cells were injected subcutaneously. 7–10 days after tumor inoculation, SFV vectors were administered intratumorally in a total volume of 50 μL diluted with saline. In the first experiment, 2 × 10 cells were injected intratumorally to compare the antitumor activity of different vectors. 7A dose of 100 viral particles / tumor was used. The following vectors were included in this experiment: SFV-scIL12-Nb11, SFV-dcIL12-Nb11, and SFV-dcIL12-bis. For comparison with those expressing ICK, SFV-scIL12 and SFV-dcIL12 were also included. Control untreated mice were administered the same volume of saline. As can be seen in Figures 14A and 14B, SFV vectors with Nb11 fused to either scIL12 or dcIL12 showed a high antitumor response, resulting in long-term survival rates of 42% and 57%, respectively. In the case of SFV-dcIL12-Nb11, the response was much higher than that of SFV-dcIL12, which only resulted in a long-term survival rate of 12.5%. For SFV-scIL12-Nb11, the response was similar to that of SFV-scIL12, but the first vector was able to better control tumor growth (Figure 14A). Serum IL-12 levels were also measured 24 hours after treatment. Most mice did not exhibit circulating IL-12, and extremely low levels were detected in four mice, each belonging to a different group (Figure 14C). No toxicity was observed in any group, as no weight loss was detectable (Figure 14D).

[0200] Because SFV-dcIL12-Nb11 showed remarkable antitumor activity, a dose-finding study was performed in comparison with SFV-dcIL12. For this purpose, 2 × 10 6 , 1×10 7 and 5 × 10 7 Three doses of 1 x 10 viral particles were used to treat MC38 tumors as described above. As shown in Figures 15A and 15B, SFV-dcIL12-Nb11 was more effective than SFV-dcIL12 at all doses tested. For example, at 1 x 10 of SFV-dcIL12-Nb11, 7 5 x 10 VP of SFV-dcIL12 7 1 × 10 SFV-dcIL12-Nb11 induced a similar tumor growth inhibitory effect compared to 1 × 10 SFV-dcIL12-Nb11 (Figure 15A). 7The individual VPs resulted in 28% long-term surviving mice compared to 0% with the same dose of SFV-dcIL12.

[0201] List of citations - Rodriguez-Madoz JR, Prieto J, Smerdou C. Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas. Mol Ther. 2005 Jul;12(1):153-63. doi:10.1016 / j.ymthe.2005.02.011. - US Patent No. 2010 / 0028330 - Ballesteros-Briones MC, Martisova E, Casales E, Silva-Pilipich N, Bunuales M, Galindo J, Mancheno U, Gorraiz M, Lasarte JJ, Kochan G, Escors D, Sanchez-Paulete AR, Melero I, Prieto J, Hernandez-Alcoceba R, Hervas-Stubbs S, Smerdou C. Short-Term Local Expression of a PD-L1 Blocking Antibody from a Self-Replicating RNA Vector Induces Potent Antitumor Responses. Mol Ther. 2019 Nov 6;27(11):1892-1905. doi:10.1016 / j.ymthe.2019.09.016. Epub 2019 Sep 16. -Quetglas JI,Fioravanti J,Ardaiz N,Medina-Echeverz J,Baraibar I,Prieto J,Smerdou C,Berraondo P.A Semliki forest virus vector engineered to express IFNα induces efficient elimination of established tumors.Gene Ther.2012 Mar;19(3):271-8.doi:10.1038 / gt.2011.99.Epub 2011 Jul 7. -Berglund P,Smerdou C,Fleeton MN,Tubulekas I,Liljestrom P.Enhancing immune responses using suicidal DNA vaccines.Nat Biotechnol.1998 Jun;16(6):562-5.doi:10.1038 / nbt0698-562. -Liljestrom P,Garoff H.Expression of proteins using Semliki Forest virus vectors.Curr Protoc Mol Biol.2001 -Smerdou C,Liljestrom P.Two-helper RNA system for production of recombinant Semliki forest virus particles.J Virol.1999;73:1092-8 -Silva-Pilipich N,Martisova E,Ballesteros-Briones MC,Hervas-Stubbs S,Casares N,Gonzalez-Sapienza G,et al.Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity without Toxicity.Biomedicines.2020;8(12):562 -Dobin A,Davis CA,Schlesinger F,Drenkow J,Zaleski C,Jha S,et al.STAR:ultrafast universal RNA-seq aligner.Bioinformatics.2013;29:15-21 -Silva-Pilipich N,Martisova E,Ballesteros-Briones MC,Hervas-Stubbs S,Casares N,Gonzalez-Sapienza G,et al.Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity without Toxicity.Biomedicines.2020;8(12):562 -Liao Y,Smyth GK,Shi W.featureCounts:an efficient general purpose program for assigning sequence reads to genomic features.Bioinformatics.2014;30:923-30. -Harrow J,Frankish A,Gonzalez JM,Tapanari E,Diekhans M,Kokocinski F,et al.GENCODE:The reference human genome annotation for The ENCODE Project.Genome Res.2012;22:1760-74 -Gentleman RC,Carey VJ,Bates DM,Bolstad B,Dettling M,Dudoit S,et al.Bioconductor:open software development for computational biology and bioinformatics.Genome Biol.2004;5:R80. -Robinson MD,McCarthy DJ,Smyth GK.edgeR:a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics.2010;26:139-40 -Ritchie ME,Phipson B,Wu D,Hu Y,Law CW,Shi W,et al.limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res.2015;43:e47-e47. -Subramanian A,Tamayo P,Mootha VK,Mukherjee S,Ebert BL,Gillette MA,et al.Gene set enrichment analysis:A knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci.2005;102:15545-50. -Silva-Pilipich N,Lasarte-Cia A,Lozano T,Martin-Otal C,Lasarte JJ,Smerdou C.Intratumoral electroporation of a self-amplifying RNA expressing interleukin-12 induces antitumor effects in mouse models of cancer.Molecular Therapy Nucleic Acids,2022 Sep 13;29:387-399.Published online 2022 Jul 20.doi:10.1016 / j.omtn.2022.07.020. -Xiang Z,Li J,Zhang Z,Cen C,Chen W,Jiang B,et al.Comprehensive Evaluation of Anti-PD-1,Anti-PD-L1,Anti-CTLA-4 and Their Combined Immunotherapy in Clinical Trials:A Systematic Review and Meta-analysis.Front Pharmacol.2022;13:883655. -Sun G,Liu H,Shi X,Tan P,Tang W,Chen X,et al.Treatment of patients with cancer using PD-1 / PD-L1 antibodies:Adverse effects and management strategies(Review).Int J Oncol.2022;60. -Needleman and Wunsch,J.Mol.Biol.48:443-453,1970.DOI:10.1016 / 0022-2836(70)90057-4. -Altschul et al.,“Basic local alignment search tool”,1990,J.Mol.Biol,v.215,pages 403-410,1990.DOI:10.1016 / S0022-2836(05)80360-2. -Grasselly C,Denis M,Bourguignon A,Talhi N,Mathe D,Tourette A,et al.The Antitumor Activity of Combinations of Cytotoxic Chemotherapy and Immune Checkpoint Inhibitors Is Model-Dependent.Front Immunol [Internet].2018;9.Available from:https: / / www.frontiersin.org / article / 10.3389 / fimmu.2018.02100 / full -Ngiow SF,Young A,Jacquelot N,Yamazaki T,Enot D,Zitvogel L,et al.A Threshold Level of Intratumor CD8+T-cell PD1 Expression Dictates Therapeutic Response to Anti-PD1.Cancer Res [Internet].2015;75:3800-11.Available from:https: / / aacrjournals.org / cancerres / article / 75 / 18 / 3800 / 606326 / A-Threshold-Level-of-Intratumor-CD8-T-cell-PD1 -Komdeur FL,Singh A,van de Wall S,Meulenberg JJM,Boerma A,Hoogeboom BN,et al.First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers.Mol Ther.2021;29:611-25. -Markham A.Envafolimab:First Approval.Drugs.2022;82:235-40. -G.J.Lieschke,P.K.Rao,M.K.Gately,R.C.Mulligan,Bioactive murine and human interleukin-12 fusion proteins which retain antitumor activity in vivo.,Nat.Biotechnol.15(1997)35-40.https: / / doi.org / 10.1038 / nbt0197-35.

Claims

1. The following complementarity determining regions (CDRs): (i) a CDR1 described by SEQ ID NO:1, a CDR2 described by SEQ ID NO:2, and a CDR3 described by SEQ ID NO:3, or (ii) a CDR1 described by SEQ ID NO:5, a CDR2 described by SEQ ID NO:6, and a CDR3 described by SEQ ID NO:7 A single domain antibody (VHH or nanobody) comprising:

2. (i) an anti-PD-L1 Nanobody comprising a CDR1 described by SEQ ID NO: 1, a CDR2 described by SEQ ID NO: 2, and a CDR3 described by SEQ ID NO: 3, and which is capable of inhibiting the binding of PD-L1 to PD-1, or (ii) an anti-PD-1 Nanobody comprising a CDR1 described by SEQ ID NO:5, a CDR2 described by SEQ ID NO:6, and a CDR3 described by SEQ ID NO:7, wherein the nanobody is capable of inhibiting the binding of PD-L1 to PD-1; The nanobody of claim 1.

3. 3. The nanobody of claim 1, which blocks the binding of PD-L1 to PD-1 with a median inhibitory concentration (IC50) of 6 nM or less.

4. (i) a sequence set forth in SEQ ID NO: 4, or a sequence having at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% identity to SEQ ID NO: 4; or (ii) a sequence set forth in SEQ ID NO: 8, or a sequence having at least 90%, or at least 95%, or at least 98%, or at least 99%, or 100% identity to SEQ ID NO: 8; 4. The Nanobody according to any one of claims 1 to 3, comprising or consisting of:

5. A nanobody conjugate comprising a nanobody according to any one of claims 1 to 4.

6. 6. A nanobody conjugate according to claims 1 to 5, comprising at least one nanobody according to any one of claims 1 to 4 and an effector molecule.

7. 7. The nanobody conjugate of any one of claims 5 to 6, comprising two nanobodies as defined in any one of claims 1 to 4 fused to an immunoglobulin Fc domain.

8. 7. The nanobody conjugate of any one of claims 5 to 6, comprising at least one nanobody of any one of claims 1 to 4 and IL-12.

9. 9. A polynucleotide encoding a Nanobody as defined in any one of claims 1 to 4, or encoding a Nanobody conjugate as defined in any one of claims 5 to 8.

10. An expression vector comprising a polynucleotide as defined in claims 1 to 9.

11. The expression vector of claims 1 to 10, which is a replication-deficient alphavirus vector based on Semliki Forest Virus (SFV).

12. A host cell comprising a polynucleotide as defined in claim 9 or an expression vector as defined in any one of claims 10-11.

13. A viral particle comprising the expression vector of any one of claims 10 to 11 and SFV capsid and envelope proteins.

14. 14. A pharmaceutical composition comprising a Nanobody as defined in any one of claims 1 to 4, or a Nanobody conjugate as defined in any one of claims 5 to 8, or an expression vector as defined in any one of claims 10 to 11, a host cell as defined in claim 12, or a viral particle as defined in claim 13, together with a pharmaceutically acceptable excipient and / or carrier.

15. A nanobody as defined in any one of claims 1 to 4, or a nanobody conjugate as defined in any one of claims 5 to 8, or an expression vector as defined in any one of claims 10 to 11, or a host cell as defined in claim 12, or a viral particle as defined in claim 13, or a pharmaceutical composition as defined in claim 14, for use in therapy, in particular for the prevention and / or treatment of cancer.

Images