Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 16-35 are pending and being acted upon in this Office Action.
Priority
Receipt is acknowledged of papers submitted under 35 U.S.C. 119(a)-(d), which papers have been placed of record in the file.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on July 16, 2024 have been considered by the examiner and an initialed copy of the IDS is included with this Office Action.
The listing of references in the specification at pages 43-45 is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The drawings were received on May 17, 2024. These drawings are acceptable.
Specification
The disclosure is objected to because of the following informalities: Figures 1A-1B contain sequences which are not identified by SEQ ID NO: either in the figure or in the Brief Description of the Figures. Applicant is required to either amend the Figures with the corresponding Sequence Identifiers or alternatively Applicant may amend the Brief Description of the Figures with the corresponding Sequence Identifiers. See 37 C.F.R. § 1.821-1.825.
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant's cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim Objection
Claim 16 is objected to because of the following informality: “A VHH antibody recognizing the SARS-CoV-2 spike protein S1 domain, particular the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain” should have been “An isolated VHH antibody recognizing a receptor-binding domain (RBD) of a SARS-CoV-2 spike protein S1 domain wherein the VHH comprising…”.
Claim 22 is objected to because of the following informalities:
“VHH antibody …comprising a VHH sequence as shown …or a VHH antibody, which is a variant thereof” should have been “VHH antibody comprising the sequence as shown in SEQ ID NO: …or a variant thereof”.
“SEQ.” should have been “SEQ”.
Claim 25 is objected to because of the following informality: “The VHH antibody or claim 16, which is covalently or non-covalently conjugated to a heterologous moiety…oligonucleotide” should have been “A conjugate VHH antibody of claim 16, wherein the VHH is covalently or non-covalently conjugated to a heterologous moiety selected from the group consisting of a label, an effector, a fluorescence, a biotin, a peroxidase, a phosphatase, a luciferase, a hapten, an affinity tag, and an oligonucleotide”.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 16, 18-20, 24-25, 30 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Regarding claims 16, 19, 20, 30, the phrase “particularly” renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claims 18-20, the phrase “or less” renders the claims indefinite because metes and bound of “less” cannot be determined.
Regarding claims 19, 20, 24, 25, the phrase "e.g." renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claim 21, the metes and bounds of claim 21 renders vague and indefinite because the claim missing a period. One of ordinary skill in the art would not be reasonably apprised of the scope of the invention. See MPEP § 2173.05(d). MPEP § 608.01(n). All claims should end with a period.
Regarding claim 25, the phrase "such as" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 25 recites a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) is considered indefinite, since the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In this case:
claim 25 recites the broad limitation of “enzyme”, and the claim also recites “peroxidase, phosphatase or luciferase” which is a narrower statement of the range/limitation;
claim 25 recites the broad limitation of “nucleic acid”, and the claim also recites “an oligonucleotide” which is a narrower statement of the range/limitation.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 25 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 25 fails to limit the parent claim from which it depends as the parent claim 16 is drawn to a VHH antibody, whereas claim 25 is VHH antibody covalently or non-covalently conjugated to any heterologous moiety, e.g., a labeling group, a capture group or an effector group, wherein the heterologous moiety is particularly selected from a fluorescence group, biotin, an enzyme such as peroxidase, phosphatase or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 16-35 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Written Description Guidelines for examination of patent applications indicates, “the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical characteristics and/or other chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show applicant was in possession of the claimed genus.” (see MPEP 2163).
Claim 16 encompasses a VHH antibody recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain comprising
(a) a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ. ID NO: 38- 40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112, or
(b) a combination of CDR1, CDR2 and CDR3 sequences which has an identity of at least at least 90% or at least 95% to a combination of CDR1, CDR2 and CDR3 sequences of (a).
Claim 17 encompasses the VHH antibody according to claim 16 comprising a VH sequence as shown in SEQ. ID NO: 37, 17, 21, 25, 29, 33, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, or 109, or a sequence which has an identity of at least 85%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a).
Claim 18 encompasses the VHH antibody of claim 16, which binds to the RBD of the SARS-CoV-2 Wuhan strain (comprised in SEQ. ID NO: 113) and to a tetramutant RBD of a SARS-CoV-2 having the mutations K417T, L452R, E484K and N501Y (comprised in SEQ. ID NO: 114) with Preliminary Amendment an affinity expressed as dissociation constant KD of about 1000 pM or less, of about 500 pM or less, of about 100 pM or less or of about 50 pM or less.
Claim 19 encompasses the VHH antibody of claim 16, which neutralizes SARS-CoV-2, e.g., the SARS-CoV-2 Wuhan strain, and/or a SARS-CoV-2 variant, particularly a variant of concern including Alpha (N501Y), Beta (N501Y, E484K, K417N), Gamma (N501Y, E484K, K417T), Delta (L452R, T478K), Epsilon (L452R), Mu (E484K, N501Y), and/or Omicron, e.g. Omicron variant BA.2.75, at a concentration of about 5 nM or less, of about 1000 pM or less, of about 500 pM or less, of about 170 pM or less, or of about 100 pM or less.
Claim 20 encompasses the VHH antibody of claim 19, which cross-neutralizes the SARS-CoV-2 Wuhan strain, the Alpha variant, the Beta variant, the Gamma variant, the Delta variant, and the Mu variant and optionally an Omicron variant, e.g. Omicron variant BA.2.75, particularly at a concentration of about 5 nM or less, of about 1000 pM or less, of about 500 pM or less, of about 170 pM or less, or of about 100 pM or less.
Claim 21 encompasses the VHH antibody of claim 16, which has a melting temperature of at least about 65°C, of at least about 80°C, of at least 90°C or of at least about 95°C when measured under non-reducing conditions, and/or which has an aggregation temperature of at least about 50°C, of at least about 60°C, of at least 70°C or of at least about 80°C when measured under non-reducing conditions.
Claim 22 encompasses the VHH antibody of claim 16, which is selected from:
VHH antibody Re32D03 comprising a VHH sequence as shown in SEQ. ID NO: 37 or a VHH antibody, which is a variant thereof,
(ii) VHH antibody Re32E06 comprising a VHH sequence as shown in SEQ. ID NO: 105 or a VHH antibody, which is a variant thereof,
(iii) VHH antibody Re30D01 comprising a VHH sequence as shown in SEQ. ID NO: 89 or a VHH antibody, which is a variant thereof,
(iv) VHH antibody Re32B 11 comprising a VHH sequence as shown in SEQ. ID NO: 45 or a VHH antibody, which is a variant thereof, and
(v) VHH antibody Ma2H07 comprising a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof.
Claim 23 encompasses the VHH antibody of claim 16, which is a monomeric VHH antibody.
Claim 24 encompasses the VHH antibody of claim 16, which is a multimeric VHH antibody, e.g., a homotrimeric VHH antibody or a heterodimeric VHH antibody.
Claim 25 encompasses the VHH antibody of claim 16, which is covalently or non-covalently conjugated to a heterologous moiety, e.g., a labeling group, a capture group or an effector group, wherein the heterologous moiety is particularly selected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.
Claim 26 encompasses a set of two or more different VHH antibodies recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain, comprising at least one VHH antibody of claim 16.
Claim 27 encompasses a pharmaceutical composition comprising at least one VHH antibody of claim 16 and a pharmaceutically acceptable carrier or excipient.
Claim 28 encompasses the pharmaceutical composition of claim 27, formulated for administration orally, nasally or intrapulmonary.
Claim 29 encompasses the pharmaceutical composition of claim 27, formulated as an aerosol for administration by inhalation.
Claim 30 encompasses a method for the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-CoV-2, comprising administering an effective dose of the VHH antibody of claim 16, to a subject in need thereof, particularly to a human subject.
Claim 31 encompasses the method of claim 30, comprising administering an effective dose of a set of two or more different VHH antibodies.
Claim 32 encompasses the method of claim 30, wherein the disorder is associated with a SARS- CoV-2 escape mutant.
Claim 33 encompasses the method of claim 30, wherein the disorder is COVID-19.
Claim 34 encompasses the method of claim 30, wherein the VHH antibody, or the set is administered orally, nasally or intrapulmonary.
Claim 35 encompasses the method of claim 32, wherein the VHH antibody is administered by inhalation.
The specification discloses various VHH antibodies that are shown in Table 2.
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[0187] Re6H06 (a previously isolated mutation-tolerant anti-RBD VHH) bound with a KD of 3 nM to the tetramutant RBD. Ma2H07 showed with a K.sub.D of 150 pM a far better affinity. Re32D03 and Re32B11 had a low picomolar affinity with a non-detectable dissociation rate.
Example 4—Virus Infection and Neutralization Assays
[0188] Virus stocks were prepared as supernatants from Vero E6 cells infected with SARS-CoV-2 (Wuhan strain (WT with D), Alpha variant, Beta variant, Gamma variant, or Delta variant).
[0189] The virus stocks contained between 10.sup.11 and10.sup.12 copies of the virus genome per ml, as determined by reverse transcription and quantitative PCR (qRT-PCR) according to a standard protocol (Corman et al., 2020). Approximately 107 virus copies were diluted in 100 ul of cell culture media, DMEM/2% FBS, in the presence or absence of varying concentrations of the VHH antibodies under study. After 60 min incubation at 37° C., this was added to a monolayer of Vero E6 cells, i.e., 5000 cells in a well of a standard 96 well cell culture plate. Note that the inoculated amount of virus was approximately 50 times higher than needed for a complete infection of a well.
[0190] Cells were fixed 48 h later with 4% paraformaldehyde, permeabilized with 0.15% Triton X100, and stained with 2 μg/ml DAPI (to locate cell nuclei) and a cocktail of fluorescently labelled VHH antibodies that recognize three non-overlapping epitopes at the SARS-COV-2 Spike protein (5 nM of Alexa Fluor 488-labeld Re7E02, Re9C07, Re9G12, and Re9H03 and 15 nM Re8H11-Atto 565 (cf. PCT/EP2021/071309) in PBS+1% BSA). Imaging was by spinning disc fluorescence microscopy. Neutralization was considered as successful if no infected cells were detectable within the well.
[0191] The results are shown in Table 1 and FIG. 3. The newly identified VHH antibodies Re32D03, Re32B11, Re30H02 and Ma2H07 are highly potent in preventing infection by any of the four tested SARS-COV-2 strains. VHHs Re5D06R15, Re6H06 and Re9B09 have been described earlier (cf. PCT/EP2021/071309) and have been tested here in parallel. They required higher concentrations for neutralization, in particular for the Alpha, Beta, Gamma, and Delta variants. Table 1 lists lowest neutralization concentrations averaged from up to nine sets of experiments.
Example 5—Thermostability Measurement of VHH Antibodies
[0192] VHH antibodies were subjected to Differential scanning fluorimetry (DSF), which exploits that thermal unfolding exposes aromatic/hydrophobic residues, which then bind and enhance fluorescence of the added SYPRO orange dye. Assays were performed in a volume of 20 ul, at 1 mg/ml VHH concentration in 50 mM Tris/HCl, 150 mM NaCl (pH 8.0 at 20° C.) and 1× dye (diluted from a 5000× stock; Life Technologies). Two replicates of each sample were pipetted in a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® ‘B’ Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and placed onto a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were incubated for 5 min at 25° C. and then the temperature was increased in 1° C. increments of 45 seconds to 95° C. Fluorescence was measured at the end of each step with 532 nm excitation and a 555 nm long pass filter. Melting temperatures are defined as the inflection point of the first melting peak.
[0193] The results are shown in Table 1 and FIG. 4.
[0194] VHH antibody Re9B09 that was produced in reducing cytosol and hence lacks disulphide bonds melts already at 35° C., while VHH antibodies Re30H02, Re32D03 and Re32B11 showed only a negligible melting amplitude and thus remained stable throughout 95° C.
Example 6—Resistance to Heat-Induced Aggregation
[0195] VHH antibodies were diluted to a concentration of 1 mg/ml in 50 mM Tris/HCl PH 8.0, 150 mM NaCl. One set of samples was supplemented with 0.2% Tween 80 (polysorbate 80). 40ul were heated for 10 minutes to 95° C. Samples were then centrifuged at 21 000 g for 15 minutes, and soluble in precipitated fractions were analyzed by SDS-PAGE followed by Coomassie-staining.
[0196] The results are shown in Table 1 and FIG. 5.
[0197] VHH antibody Re32B11 was fully aggregation resistant. The small extent of heat-induced aggregation of Re30H02 and Re32D03 could be fully suppressed by the addition of Tween 80.
Example 7—Impact of Nebulization on the Activity
[0198] VHH antibody was diluted to a concentration of 30 mg/ml (“Solution A”). The solution was put in a nebulizer, and the aerosol was collected into a fluid again (“Solution I”).
[0199] A dilution series of both was prepared, and a virus neutralization assay was performed with the respective solutions. The results are shown in FIG. 7. A less-than-three-fold loss in activity was found upon nebulization and re-collection. This is very acceptable and encouraging for aerosol-based nanobody therapy.
Regarding sequences having at least 85%, 90% or 95% sequence identity to a combination of CDR1, CDR2 and CDR3 as shown in SEQ. ID NO: 38- 40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112 (claims 16(b), 17), the specification does not teach where and what amino acids within the full-length sequence of CDRs 1-3 above to be substituted, deleted, added or a combination thereof such that the modified VHH having at least 85% or 90% sequence identity still maintains binding to SARS-CoV-2 spike protein S1 domain or the receptor binding domain or Wuhan strain comprises SEQ ID NO: 113 or tetramutant having K417T, L452R, E484K and N501Y comprises in SEQ ID NO: 114 with the corresponding affinity expressed as dissociated constant KD of about 1000 pM or less (claims 18-19) or alpha variant (N501Y), beta (N501Y, E484K, K417N), gamma (N501Y, E484K, K417T), delta (L452R, T478K), epsilon (L452), Mu (E484K, N501Y), and/or Omicron, Omicron variant BA.2.7.5 at a concentration of about 5 nM or less (claims 19-20).
Regarding any variants of SEQ ID NO: 37, 105, 89, 45 or 25 (claim 22), other than the specific VHH comprising the amino acid sequence of SEQ ID NO: 37, 17, 21, 33, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105 and 109, the specification has not shown which of the amino acid residues in each of the SEQ ID NO above can be mutated for a variant VHH that would maintain the same functions. These claims lack written description because the specification lacks a representative number of species that satisfies the entirety of the genus.
It was known in the art that single amino acid substitutions in VHH CDRs can alter antigen binding, stability, and developability, but their effects are often unpredictable due to complex sequence–structure–function relationships and species-specific patterns.
For example, Erasmus (MABS 17(1): 2516676, 2025; PTO 892) teaches the most common method for generating therapeutic VHHs involves immunization, followed by humanization and further optimization to enhance affinity and developability. However, many VHH advantages tend to be reduced or eliminated when VHHs are humanized by replacing key framework residues with corresponding amino acids from the most similar human germlines, requiring extensive further optimization for VHHs used as therapeutics. As an alternative approach, naïve VHH display libraries have been created. These fall into two categories: 1) those derived from naturally occurring, non-immunized camelid VHHs 32–35 which also require subsequent humanization, and 2) more advanced synthetic libraries. 36–41 A primary challenge in using naturally occurring VHHs for therapeutic applications, whether from immunization or display libraries, lies in the need for downstream optimization after discovery. Despite their similarity to human antibodies, VH3–23 in particular, camelid VHHs are routinely humanized to ensure their sequences more closely resemble those of human molecules, with subsequent optimization to maintain solubility, affinity, stability, immunogenicity profiles and “VHH-like” characteristics. In addition to humanization and aggregation challenges, downstream purification is another significant obstacle in therapeutic VHH development, see p. 2, left col. in particular. Erasmus further teaches that while a commonly used rationale for using VHHs, particularly camelid-based, relates to their longer HCDR3s, confer ring unique properties in binding different surfaces (convex, concave, and flat), 25 we have found this length characteristic is not typical for therapeutic VHHs (Figure 2l). Importantly, because it is known longer HCDR3s elevate the risk of poly reactivity, the use of longer camelid CDR3s may complicate the development of therapeutic VHHs. Furthermore, longer CDR3s often require disulfide stabilization 57 to other CDRs (e.g., CDR1/2, from which we have removed cysteines), and are more relevant in the context of camelid immune systems, not VHH therapeutics, see p. 11, right col.
De Genst et al. (Developmental and Comparative Immunology 30: 187-198. 2006; PTO 892) teaches that the VHH amino acid sequences resembles closely to that of a human VH of family III, with notable differences in its FR2 and CDRs in that the hydrophobic amino acid residues in the human VH FR2 region (e.g. the highly conserved Val42) was replaced with more hydrophilic resides in VHH (Phe42) and the conserved hydrophobic amino acid residues in the VHH FR2 region are important in maintaining the solubility of the isolated VHH domain (e.g. see page 188). De Genst et al. further teach that since the three antigen binding loops provided by a VL are absent in the antigen-binding site in the VHH, the three CDRs of the VHH are critical for antigen binding specificity and affinity and that cloning VHH antibody (e.g. by phage display or camelizing CDR3 by using a synthetic library) can be disadvantageous since lower affinity binders are often obtained that need additional mutagenesis steps (e.g. see right column on page 188 and left column on page 196).
Regarding prevention or treatment of disorder associated with SARS-CoV-2 (claims 30-35), one cannot extrapolate the teaching of the specification to the claims because it is well known that the art of epitope resilience in the face of emerging SARS-CoV-2 variants of concern (VOCs) is a key concern for antibody-based antiviral agents, with both casirivimab (REGN10933; Regeneron) and bamlanivimab (LyCo555; Eli Lilly) reported to lose activity against B.1.351.
For example, Wheatley (Cell Reports 37: 109822, Oct 2021; PTO 892) teaches SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, thegenerationofadiversepanelof91human,neutralizingmonoclonalantibodiesprovidesanin-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1, abstract, in particular. The utility of combining two or more antibodies into cocktails to limit the selection of viral escape mutants has been established in vitro. Now, in the context of emerging VOCs, antibody cocktails also provide a logical strategy for redundant protection in the face of ongoing viral evolution. However, the parameters for optimal cocktail selection remain unclear, see p. 8, right col. Wheatley teaches the loss of recognition and/or neutralizing activity against B.1.351 and P.1 VOCs for nearly one-half of the mAbs tested. The effect was greatest for the widely prevalent VH3-53/66 class of RBD-specific antibodies and for NTD-specific mAbs in which the deletions and mutations reported in VOCs appear to alter the structural conformation of this domain.
There are insufficient in vivo working examples of preventing SARS-CoV-2 infection. It is unpredictable which undisclosed VHH variants are effective for treating, much less preventing any and all disorder caused by and/or associated with an infection with SARS-CoV-2 variants in any human subject.
“Adequate written description requires a precise definition, such as by structure, formula, chemical name or physical properties, not a mere wish or plan for obtaining the claimed chemical invention.” Regents of the University of California v. Eli Lilly and Co. 43 USPQ2d 1398 (Fed. Cir. 1997).
The disclosure must allow one skilled in the art to visualize or recognize the identity of the subject matter of the claim. Id. 43 USPQ2d at 1406.
In the absence of disclosure of relevant, identifying amino acid within the CDRs of the VHH having any modification such as addition, deletion, substitution and a combination thereof that has no resemblance to the CDRs still maintains binding, the claims are not adequately described to demonstrate possession at the time of filing.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the written description inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116.).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence. Thus, the specification fails to describe these DNA sequences.
For genus claims, an adequate written description of a claimed genus requires more than a generic statement of an invention's boundaries. A patent must set forth either a representative number of species falling within the scope of the genus or structural features common to the members of the genus. Kubin, Exparte, 83 USPQ2d 1410 (Bd. Pat. App. & Int. 2007); Ariad Pharms., Inc. v. Eli Lilly& Co., 598 F.3d 1336, 1350 (Fed. Cir. 2010).
A description adequate to satisfy 35 U.S.C. § 112, first paragraph, must ‘“clearly allow persons of ordinary skill in the art to recognize that [the inventor] invented what is claimed.’” Ariad Pharms., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1351 (Fed. Cir. 2010) (enbanc) (citation omitted, alteration in original). “[T]he test for sufficiency is whether the disclosure of the application relied upon reasonably conveys to those skilled in the art that the inventor had possession of the claimed subject matter as of the filing date.” Id.
Therefore, only (1) an isolated VHH antibody recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 Sl domain comprising a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 38-40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112, (2) The isolated VHH comprising the amino acid sequence as set forth in in SEQ ID NO: 37, 17, 21, 33, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, or 109 or a conservative variant thereof wherein the variant is at least 95% sequence identity to any one of said VHH, but not the full breadth of the claims meets the written description provision of 35 U.S.C. § 112, first paragraph.
Claims 16-35 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for (1) an isolated VHH antibody recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 Sl domain comprising a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 38- 40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112, (2) The isolated VHH comprising the amino acid sequence as set forth in in SEQ ID NO: 37, 17, 21, 33, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, or 109 or a conservative variant thereof wherein the variant is at least 95% sequence identity to any one of said VHH, does not reasonably provide enablement for any and all VHH as set forth in claims 16-29 for prevention of any disorder caused by and/or associated with SARS-CoV-2 as set forth in claims 30-35. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). These factors include, but are not limited to: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. . In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988).
Claim 16 encompasses a VHH antibody recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 Sl domain comprising
(a) a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ. ID NO: 38- 40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112, or
(b) a combination of CDR1, CDR2 and CDR3 sequences which has an identity of at least at least 90% or at least 95% to a combination of CDR1, CDR2 and CDR3 sequences of (a).
Claim 17 encompasses the VHH antibody according to claim 16 comprising a VH sequence as shown in SEQ. ID NO: 37, 17, 21, 25, 29, 33, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, or 109, or a sequence which has an identity of at least 85%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a).
Claim 18 encompasses the VHH antibody of claim 16, which binds to the RBD of the SARS-CoV-2 Wuhan strain (comprised in SEQ. ID NO: 113) and to a tetramutant RBD of a SARS-CoV-2 having the mutations K417T, L452R, E484K and N501Y (comprised in SEQ. ID NO: 114) with Preliminary Amendment an affinity expressed as dissociation constant KD of about 1000 pM or less, of about 500 pM or less, of about 100 pM or less or of about 50 pM or less.
Claim 19 encompasses the VHH antibody of claim 16, which neutralizes SARS-CoV-2, e.g., the SARS-CoV-2 Wuhan strain, and/or a SARS-CoV-2 variant, particularly a variant of concern including Alpha (N501Y), Beta (N501Y, E484K, K417N), Gamma (N501Y, E484K, K417T), Delta (L452R, T478K), Epsilon (L452R), Mu (E484K, N501Y), and/or Omicron, e.g. Omicron variant BA.2.75, at a concentration of about 5 nM or less, of about 1000 pM or less, of about 500 pM or less, of about 170 pM or less, or of about 100 pM or less.
Claim 20 encompasses the VHH antibody of claim 19, which cross-neutralizes the SARS-CoV-2 Wuhan strain, the Alpha variant, the Beta variant, the Gamma variant, the Delta variant, and the Mu variant and optionally an Omicron variant, e.g. Omicron variant BA.2.75, particularly at a concentration of about 5 nM or less, of about 1000 pM or less, of about 500 pM or less, of about 170 pM or less, or of about 100 pM or less.
Claim 21 encompasses the VHH antibody of claim 16, which has a melting temperature of at least about 65°C, of at least about 80°C, of at least 90°C or of at least about 95°C when measured under non-reducing conditions, and/or which has an aggregation temperature of at least about 50°C, of at least about 60°C, of at least 70°C or of at least about 80°C when measured under non-reducing conditions.
Claim 22 encompasses the VHH antibody of claim 16, which is selected from:
VHH antibody Re32D03 comprising a VHH sequence as shown in SEQ. ID NO: 37 or a VHH antibody, which is a variant thereof,
(ii) VHH antibody Re32E06 comprising a VHH sequence as shown in SEQ. ID NO: 105 or a VHH antibody, which is a variant thereof,
(iii) VHH antibody Re30D01 comprising a VHH sequence as shown in SEQ. ID NO: 89 or a VHH antibody, which is a variant thereof,
(iv) VHH antibody Re32B 11 comprising a VHH sequence as shown in SEQ. ID NO: 45 or a VHH antibody, which is a variant thereof, and
(v) VHH antibody Ma2H07 comprising a VHH sequence as shown in SEQ. ID NO: 25 or a VHH antibody, which is a variant thereof.
Claim 23 encompasses the VHH antibody of claim 16, which is a monomeric VHH antibody.
Claim 24 encompasses the VHH antibody of claim 16, which is a multimeric VHH antibody, e.g., a homotrimeric VHH antibody or a heterodimeric VHH antibody.
Claim 25 encompasses the VHH antibody of claim 16, which is covalently or non-covalently conjugated to a heterologous moiety, e.g., a labeling group, a capture group or an effector group, wherein the heterologous moiety is particularly selected from a fluorescence group, biotin, an enzyme such as a peroxidase, phosphatase or luciferase, a hapten, an affinity tag, or a nucleic acid such as an oligonucleotide.
Claim 26 encompasses a set of two or more different VHH antibodies recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain, comprising at least one VHH antibody of claim 16.
Claim 27 encompasses a pharmaceutical composition comprising at least one VHH antibody of claim 16 and a pharmaceutically acceptable carrier or excipient.
Claim 28 encompasses the pharmaceutical composition of claim 27, formulated for administration orally, nasally or intrapulmonary.
Claim 29 encompasses the pharmaceutical composition of claim 27, formulated as an aerosol for administration by inhalation.
Claim 30 encompasses a method for the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-CoV-2, comprising administering an effective dose of the VHH antibody of claim 16, to a subject in need thereof, particularly to a human subject.
Claim 31 encompasses the method of claim 30, comprising administering an effective dose of a set of two or more different VHH antibodies.
Claim 32 encompasses the method of claim 30, wherein the disorder is associated with a SARS- CoV-2 escape mutant.
Claim 33 encompasses the method of claim 30, wherein the disorder is COVID-19.
Claim 34 encompasses the method of claim 30, wherein the VHH antibody, or the set is administered orally, nasally or intrapulmonary.
Claim 35 encompasses the method of claim 32, wherein the VHH antibody is administered by inhalation.
Enablement is not commensurate in scope with claims as how to use any isolated human antibody or binding fragment thereof mentioned above wherein said antibody or fragment thereof comprises just heavy chain amino acid sequence of antibody 131 (SEQ ID NO: 2) or just the light chain amino acid sequence of antibody 131 (SEQ ID NO: 19) or just the heavy chain CDRs 1-3 within SEQ ID NO: 2 or just the light chain CDRs 1-3 within SEQ ID NO: 19 for treating any mammal such as human suffers from any cancer involving epithelial cell proliferation, any lung cancer, any colon cancer, any gastric cancer, renal cancer, prostate cancer, breast cancer, glioblastoma or ovarian carcinoma.
The specification discloses various VHH antibodies that are shown in Table 2.
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[0187] Re6H06 (a previously isolated mutation-tolerant anti-RBD VHH) bound with a KD of 3 nM to the tetramutant RBD. Ma2H07 showed with a K.sub.D of 150 pM a far better affinity. Re32D03 and Re32B11 had a low picomolar affinity with a non-detectable dissociation rate.
Example 4—Virus Infection and Neutralization Assays
[0188] Virus stocks were prepared as supernatants from Vero E6 cells infected with SARS-CoV-2 (Wuhan strain (WT with D), Alpha variant, Beta variant, Gamma variant, or Delta variant).
[0189] The virus stocks contained between 10.sup.11 and10.sup.12 copies of the virus genome per ml, as determined by reverse transcription and quantitative PCR (qRT-PCR) according to a standard protocol (Corman et al., 2020). Approximately 107 virus copies were diluted in 100 ul of cell culture media, DMEM/2% FBS, in the presence or absence of varying concentrations of the VHH antibodies under study. After 60 min incubation at 37° C., this was added to a monolayer of Vero E6 cells, i.e., 5000 cells in a well of a standard 96 well cell culture plate. Note that the inoculated amount of virus was approximately 50 times higher than needed for a complete infection of a well.
[0190] Cells were fixed 48 h later with 4% paraformaldehyde, permeabilized with 0.15% Triton X100, and stained with 2 μg/ml DAPI (to locate cell nuclei) and a cocktail of fluorescently labelled VHH antibodies that recognize three non-overlapping epitopes at the SARS-COV-2 Spike protein (5 nM of Alexa Fluor 488-labeld Re7E02, Re9C07, Re9G12, and Re9H03 and 15 nM Re8H11-Atto 565 (cf. PCT/EP2021/071309) in PBS+1% BSA). Imaging was by spinning disc fluorescence microscopy. Neutralization was considered as successful if no infected cells were detectable within the well.
[0191] The results are shown in Table 1 and FIG. 3. The newly identified VHH antibodies Re32D03, Re32B11, Re30H02 and Ma2H07 are highly potent in preventing infection by any of the four tested SARS-COV-2 strains. VHHs Re5D06R15, Re6H06 and Re9B09 have been described earlier (cf. PCT/EP2021/071309) and have been tested here in parallel. They required higher concentrations for neutralization, in particular for the Alpha, Beta, Gamma, and Delta variants. Table 1 lists lowest neutralization concentrations averaged from up to nine sets of experiments.
Example 5—Thermostability Measurement of VHH Antibodies
[0192] VHH antibodies were subjected to Differential scanning fluorimetry (DSF), which exploits that thermal unfolding exposes aromatic/hydrophobic residues, which then bind and enhance fluorescence of the added SYPRO orange dye. Assays were performed in a volume of 20 ul, at 1 mg/ml VHH concentration in 50 mM Tris/HCl, 150 mM NaCl (pH 8.0 at 20° C.) and 1× dye (diluted from a 5000× stock; Life Technologies). Two replicates of each sample were pipetted in a Hard-Shell® 96-well plate (Bio-Rad). The plate was sealed with transparent MicroSeal® ‘B’ Seal (Bio-Rad), briefly centrifuged to remove any air bubbles, and placed onto a CFX96 Real-Time System (C1000 Thermal Cycler, BioRad). The samples were incubated for 5 min at 25° C. and then the temperature was increased in 1° C. increments of 45 seconds to 95° C. Fluorescence was measured at the end of each step with 532 nm excitation and a 555 nm long pass filter. Melting temperatures are defined as the inflection point of the first melting peak.
[0193] The results are shown in Table 1 and FIG. 4.
[0194] VHH antibody Re9B09 that was produced in reducing cytosol and hence lacks disulphide bonds melts already at 35° C., while VHH antibodies Re30H02, Re32D03 and Re32B11 showed only a negligible melting amplitude and thus remained stable throughout 95° C.
Example 6—Resistance to Heat-Induced Aggregation
[0195] VHH antibodies were diluted to a concentration of 1 mg/ml in 50 mM Tris/HCl PH 8.0, 150 mM NaCl. One set of samples was supplemented with 0.2% Tween 80 (polysorbate 80). 40ul were heated for 10 minutes to 95° C. Samples were then centrifuged at 21 000 g for 15 minutes, and soluble in precipitated fractions were analyzed by SDS-PAGE followed by Coomassie-staining.
[0196] The results are shown in Table 1 and FIG. 5.
[0197] VHH antibody Re32B11 was fully aggregation resistant. The small extent of heat-induced aggregation of Re30H02 and Re32D03 could be fully suppressed by the addition of Tween 80.
Example 7—Impact of Nebulization on the Activity
[0198] VHH antibody was diluted to a concentration of 30 mg/ml (“Solution A”). The solution was put in a nebulizer, and the aerosol was collected into a fluid again (“Solution I”).
[0199] A dilution series of both was prepared, and a virus neutralization assay was performed with the respective solutions. The results are shown in FIG. 7. A less-than-three-fold loss in activity was found upon nebulization and re-collection. This is very acceptable and encouraging for aerosol-based nanobody therapy.
Regarding sequences having at least 85%, 90%, or 95% sequence identity to a combination of CDR1, CDR2 and CDR3 as shown in SEQ. ID NO: 38- 40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112 (claims 16(b), 17), the specification does not teach where and what amino acids within the full-length sequence of CDRs 1-3 above to be substituted, deleted, added or a combination thereof such that the modified VHH having at least 85%, 90% or 95% sequence identity still maintains binding to SARS-CoV-2 spike protein S1 domain or the receptor binding domain or Wuhan strain comprises SEQ ID NO: 113 or tetramutant having K417T, L452R, E484K and N501Y comprises in SEQ ID NO: 114 with the corresponding affinity expressed as dissociated constant KD of about 1000 pM or less (claims 18-19) or alpha variant (N501Y), or beta (N501Y, E484K, K417N), or gamma (N501Y, E484K, K417T), or delta (L452R, T478K), or epsilon (L452), Mu (E484K, N501Y), and/or Omicron, Omicron variant BA.2.7.5 at a concentration of about 5 nM or less (claims 19-20).
Regarding any variants of SEQ ID NO: 37, 105, 89, 45 or 25 (claim 22), other than the specific VHH comprising the amino acid sequence of SEQ ID NO: 37, 17, 21, 33, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105 and 109, the specification has not shown which of the amino acid residues in each of the SEQ ID NO above can be mutated such that the variant of VHH would maintain the same functions. These claims lack written description because the specification lacks a representative number of species that satisfies the entirety of the genus.
It was known in the art that single amino acid substitutions in VHH CDRs can alter antigen binding, stability, and developability, but their effects are often unpredictable due to complex sequence–structure–function relationships and species-specific patterns.
For example, Erasmus (MABS 17(1): 2516676, 2025; PTO 892) teaches the most common method for generating therapeutic VHHs involves immunization, followed by humanization and further optimization to enhance affinity and developability. However, many VHH advantages tend to be reduced or eliminated when VHHs are humanized by replacing key framework residues with corresponding amino acids from the most similar human germlines, requiring extensive further optimization for VHHs used as therapeutics. As an alternative approach, naïve VHH display libraries have been created. These fall into two categories: 1) those derived from naturally occurring, non-immunized camelid VHHs 32–35 which also require subsequent humanization, and 2) more advanced synthetic libraries. 36–41 A primary challenge in using naturally occurring VHHs for therapeutic applications, whether from immunization or display libraries, lies in the need for downstream optimization after discovery. Despite their similarity to human antibodies, VH3–23 in particular, camelid VHHs are routinely humanized to ensure their sequences more closely resemble those of human molecules, with subsequent optimization to maintain solubility, affinity, stability, immunogenicity profiles and “VHH-like” characteristics. In addition to humanization and aggregation challenges, downstream purification is another significant obstacle in therapeutic VHH development, see p. 2, left col. in particular. Erasmus further teaches that while a commonly used rationale for using VHHs, particularly camelid-based, relates to their longer HCDR3s, confer ring unique properties in binding different surfaces (convex, concave, and flat), 25 we have found this length characteristic is not typical for therapeutic VHHs (Figure 2l). Importantly, because it is known longer HCDR3s elevate the risk of poly reactivity, the use of longer camelid CDR3s may complicate the development of therapeutic VHHs. Furthermore, longer CDR3s often require disulfide stabilization 57 to other CDRs (e.g., CDR1/2, from which we have removed cysteines), and are more relevant in the context of camelid immune systems, not VHH therapeutics, see p. 11, right col.
De Genst et al. (Developmental and Comparative Immunology 30: 187-198, 2006; PTO 892) teaches that the VHH amino acid sequences resembles closely to that of a human VH of family III, with notable differences in its FR2 and CDRs in that the hydrophobic amino acid residues in the human VH FR2 region (e.g. the highly conserved Val42) was replaced with more hydrophilic resides in VHH (Phe42) and the conserved hydrophobic amino acid residues in the VHH FR2 region are important in maintaining the solubility of the isolated VHH domain (e.g. see page 188). De Genst et al. further teach that since the three antigen binding loops provided by a VL are absent in the antigen-binding site in the VHH, the three CDRs of the VHH are critical for antigen binding specificity and affinity and that cloning VHH antibody (e.g. by phage display or camelizing CDR3 by using a synthetic library) can be disadvantageous since lower affinity binders are often obtained that need additional mutagenesis steps (e.g. see right column on page 188 and left column on page 196).
Regarding prevention or treatment of disorder associated with SARS-CoV-2, one cannot extrapolate the teaching of the specification to the claims because it is well known that the art of epitope resilience in the face of emerging SARS-CoV-2 variants of concern (VOCs) is a key concern for antibody-based antiviral agents, with both casirivimab (REGN10933; Regeneron) and bamlanivimab (LyCo555; Eli Lilly) reported to lose activity against B.1.351.
For example, Wheatley (Cell Reports 37: 109822, Oct 2021; PTO 892) teaches SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, thegenerationofadiversepanelof91human,neutralizingmonoclonalantibodiesprovidesanin-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1, abstract, in particular. The utility of combining two or more antibodies into cocktails to limit the selection of viral escape mutants has been established in vitro. Now, in the context of emerging VOCs, antibody cocktails also provide a logical strategy for redundant protection in the face of ongoing viral evolution. However, the parameters for optimal cocktail selection remain unclear, see p. 8, right col. Wheatley teaches the loss of recognition and/or neutralizing activity against B.1.351 and P.1 VOCs for nearly one-half of the mAbs tested. The effect was greatest for the widely prevalent VH3-53/66 class of RBD-specific antibodies and for NTD-specific mAbs in which the deletions and mutations reported in VOCs appear to alter the structural conformation of this domain.
There are insufficient in vivo working examples of preventing SARS-CoV-2 infection, much less treating any and all disorders caused by SARS-Co-V-2 infection. It is unpredictable which undisclosed VHH variants are effective for preventing any and all disorder caused by and/or associated with an infection with SARS-CoV-2 variants in any human subject.
As such, it would require undue experimentation of one skilled in the art to practice the claimed invention.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 16-35 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by GÖRLICH ( US20230287087, filed January 27, 2023, claimed earliest priority to EP20188451.7 filed July 29, 2020; PTO 892).
16. (New) A VHH antibody recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 Sl domain comprising (a) a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ. ID NO: 38- 40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112, or (b) a combination of CDR1, CDR2 and CDR3 sequences which has an identity of at least at least 90% or at least 95% to a combination of CDR1, CDR2 and CDR3 sequences of (a).
17. (New) The VHH antibody according to claim 16 comprising a VH sequence as shown in SEQ. ID NO: 25, or a sequence which has an identity of at least 85%, at least 90%, at least 95% or at least 99% to a VHH sequence of (a).
Regarding claims 16 (b), and 17, GÖRLICH teaches VHH comprising the amino acid sequence of SEQ ID NO: 129 that recognizes SARS-CoV-2 spike protein S1 domain (para. [0037]), particularly the receptor-biding domain (RBD, para. [0037]) wherein the VHH comprising a combination of CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 130 to 132 which has a sequence identity of 90.3 % to the claimed SQE ID NO: 25, the three CDRs are in bold, see sequence alignment below, red arrow in Table:
Query Match 90.3%; Score 633; Length 134;
Best Local Similarity 90.8%;
Matches 118; Conservative 4; Mismatches 8; Indels 0; Gaps 0;
Qy 1 QVQLVESGGGLVQPGGSLRLSCAYSGITLDYYAIGWFRQAPGKEREGVSYISSSNDRTDY 60
||||||||||||||||||||||| || |||||||||||||||||||||| ||||: |||
Db 3 QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSRISSSDGSTDY 62
Qy 61 ADSVKGRFTISRDNAKNTVFLQMNSLQPEDTAVYYCATVTGTYYSGRWYYNWHPEAVDYW 120
|||||||||||||||||||:||||||:|||||||||||| |||||| ||| |||:|||||
Db 63 ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATVPGTYYSGNWYYTWHPKAVDYW 122
Qy 121 GKGTLVTVSS 130
||||||||||
Db 123 GKGTLVTVSS 132
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Regarding claims 18-20, GÖRLICH discloses the VHH antibody of any one of embodiments 1-6, which is capable of neutralizing a SARS-CoV2 mutant comprising a spike protein RBD including at least one amino acid substitution in the RBD selected from the group consisting of K417T, K417N, L452R, E484K, N501 and T478K, see para. [0058]. The VHH Re9B09 monomer or heterodimer has an affinity expressed as dissociation constant KD of about 50 pM or less, e.g., 10 pM to the wild type or 100 pM to the Delta variant (L452R, T478K), see Table 7, below:
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Regarding claim 21, GÖRLICH teaches the VHH antibody of embodiment 9, which has a melting temperature of at least about 65° C., of at least about 80° C., of at least 90° C. or of at least about 95° C. when measured under non-reducing conditions and/or under reducing conditions, see para. [0060].
The VHH antibody of embodiment 9 or 10, which has an aggregation temperature of at least about 50° C., of at least about 60° C., of at least 70° C. or of at least about 80° C. when measured under non-reducing conditions and/or under reducing conditions, see para. [0062].
Regarding claim 22, GÖRLICH teaches VHH variants of SEQ ID NO: 1, 5, 9, 13, 17, or 21, see VHH sequences in TABLE 2, in particular.
Regarding claims 23-24, GÖRLICH teaches that the VHH antibody is monomeric as per claim 23 or multimeric, e.g. homotrimeric VHH antibody as per claim 24 (see para. [0131], [0132]) or heterodimeric VHH antibody, see para. [0133], in particular.
Regarding claim 25, GÖRLICH teaches that the VHH antibody as described above is covalently or non-covalently conjugated to a heterologous moiety, which may be selected from a labeling group, a capture group or an effector group, or hydrophilic polymer, see para. [0016].
Regarding claim 26, GÖRLICH teaches a set of two or more different VHH antibodies as described above, wherein the different VHH antibodies may recognize different epitopes, particularly different epitopes on the RBD, and more particularly non-overlapping epitopes on the RBD, see para. [0022].
Regarding claim 27, GÖRLICH teaches typically, the VHH antibody is administered as a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments are well known in the art, see para. [0335], in particular.
Regarding claims 28-29, GÖRLICH teaches the pharmaceutical composition may be administered locally, e.g. orally, nasally or intrapulmonary, for example by inhalation as an aerosol, see para. [0337].
Regarding claims 30-31, GÖRLICH teaches a method for the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-CoV-2, comprising administering an effective dose of the VHH antibody including a heterodimeric VHH antibody as described above or the set of at least two different VHH antibodies as described above to a subject in need thereof, particularly to a human subject infected with SARS-CoV-2 or at risk from being infected with SARS-CoV-2, see para. [0025].
Regarding claim 32, GÖRLICH teaches that the VHH antibody is for use in the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-CoV-2 including an infection with a SARS-CoV-2 escape mutant, see para. [0080].
Regarding claim 33, GÖRLICH teaches that the VHH antibody is for use in the prevention or treatment of Covid-19 in a human subject, see para. [0081], in particular.
Regarding claims 34-35, GÖRLICH teaches that the VHH antibody is administered orally, nasally, or intrapulmonary, for example by inhalation as an aerosol, see para. [0337], in particular.
Thus, the reference teachings anticipate the claimed invention.
Claims 16-35 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by GÖRLICH (US20230303664, filed January 29, 2021, claimed earliest priority to EP20188451.7 filed July 29, 2020; PTO 892).
16. (New) A VHH antibody recognizing the SARS-CoV-2 spike protein S1 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 Sl domain comprising (a) a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ. ID NO: 38- 40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112, or (b) a combination of CDR1, CDR2 and CDR3 sequences which has an identity of at least at least 90% or at least 95% to a combination of CDR1, CDR2 and CDR3 sequences of (a).
17. (New) The VHH antibody according to claim 16 comprising a VH sequence as shown in SEQ. ID NO: 29, or a sequence which has an identity of at least 85% to a VHH sequence of (a).
Regarding claim 17, GÖRLICH teaches an VHH antibody recognizing the SARS-CoV-2 spike protein 51 domain, particularly the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain (see entire document, para. [0013]) wherein the VHH comprising the amino acid sequence of SEQ ID NO: 252 and wherein the VHH comprising a combination of CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 130 to 132 which has a sequence identity of 89.5 % to the claimed SEQ ID NO: 29, which is at least 85% to the claimed VHH comprising the three CDRs, see sequence alignment below and Table 2, claims in particular:
Query Match 89.6%; Score 630; Length 130;
Best Local Similarity 90.0%;
Matches 117; Conservative 4; Mismatches 9; Indels 0; Gaps 0;
Qy 1 QVQLVESGGGLVQPGGSLRLSCAYSGITLDYYAIGWFRQAPGKEREGVSYISGSNDRTDY 60
||||||||| |||| |||||||| |||||||||||||||||||||||||||| :||:|||
Db 1 QVQLVESGGDLVQPRGSLRLSCAASGITLDYYAIGWFRQAPGKEREGVSYISNTNDKTDY 60
Qy 61 ADSVKGRFTISRDNAKNTVFLQMNSLEPEDTAVYYCATVTGTYYSGRWYYNWHPEAVDYW 120
|||||||||||||||:||||||||||:|||||||||||| |||| ||||| |||||| ||
Db 61 ADSVKGRFTISRDNAENTVFLQMNSLQPEDTAVYYCATVPGTYYGGRWYYTWHPEAVRYW 120
Qy 121 GKGTLVTVSS 130
|||| |||||
Db 121 GKGTQVTVSS 130
PNG
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225
929
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Greyscale
Regarding claims 18-20, GÖRLICH discloses the SARS-CoV-2-neutralizing VHH antibody is capable of neutralizing a SARS-CoV2 mutant, in particular a SARS-CoV-2 escape mutant. In certain embodiments, the SARS-CoV-2-neutralizing VHH antibody is capable of neutralizing a SARS-CoV2 mutant comprising a spike protein RBD including at least one amino acid substitution in the RBD selected from the group consisting of K417T, K417N, L452R, E484K, and N501Y, see para. [0020].
Regarding claims 19-20, GÖRLICH discloses the VHH antibody of the present invention is binding to the 51 domain, particularly to the RBD of the S1 domain of SARS-CoV-2. In certain embodiments, a VHH antibody is capable of virus neutralization at a concentration of about 10 nM or less, of about 1 nM or less, of about 500 pM or less, of about 100 pM or less, or even at about 50 pM or less, see para. [0187].
The VHH antibody is capable of neutralizing a SARS-CoV2 mutant comprising a spike protein RBD including at least one amino acid substitution in the RBD selected from the group consisting of K417T, K417N, L452R, E484K, and N501Y. Thus, the present invention provides a VHH antibody capable of neutralizing a SARS-CoV-2 escape mutant, e.g. the UK mutant (Alpha variant, B.1.1.7), the South African mutant (Beta variant, B.1.351), the Brazilian mutant (Gamma variant, P.1), the Indian mutant (Delta variant, B.1.617) and/or the Californian mutant (Epsilon variant, B.1.427), see para. [0188].
Regarding claim 21, GÖRLICH discloses the VHH antibody, which has a melting temperature of at least about 65° C., of at least about 80° C., of at least 90° C. or of at least about 95° C. when measured under non-reducing conditions and/or under reducing conditions, see para. [0059].
The VHH antibody of embodiment 9 or 10, which has an aggregation temperature of at least about 50° C., of at least about 60° C., of at least 70° C. or of at least about 80° C. when measured under non-reducing conditions and/or under reducing conditions, see para. [0062].
Regarding claim 22, GÖRLICH discloses the VHH antibody Re9H03 comprising a VHH sequence as shown in SEQ. ID NO: 252 or a VHH antibody, which is a variant thereof, particularly a variant as shown in SEQ. ID NO: 272 (Re22E05), see para. [0063] to [0064].
Regarding claims 23-24, GÖRLICH teaches that the VHH antibody is monomeric as per claim 23 or multimeric, e.g. homotrimeric VHH antibody as per claim 24 (see para. [0128] to [0131]) or heterodimeric VHH antibody, see para. [0132], in particular.
Regarding claim 25, GÖRLICH teaches that the VHH antibody as described above is covalently or non-covalently conjugated to a heterologous moiety, which may be selected from a labeling group, a capture group or an effector group, or hydrophilic polymer, see para. [0016] to [0018].
Regarding claim 26, GÖRLICH teaches a set of two or more different VHH antibodies as described above, wherein the different VHH antibodies may recognize different epitopes, particularly different epitopes on the RBD, and more particularly non-overlapping epitopes on the RBD, see para. [0022].
Regarding claim 27, GÖRLICH teaches typically, the VHH antibody is administered as a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments are well known in the art, see para. [0335], in particular.
Regarding claims 28-29, GÖRLICH teaches the pharmaceutical composition may be administered locally, e.g. orally, nasally or intrapulmonary, for example by inhalation as an aerosol, see para. [0337].
Regarding claims 30-31, GÖRLICH teaches a method for the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-CoV-2, comprising administering an effective dose of the VHH antibody including a heterodimeric VHH antibody as described above or the set of at least two different VHH antibodies as described above to a subject in need thereof, particularly to a human subject infected with SARS-CoV-2 or at risk from being infected with SARS-CoV-2, see para. [0022], [0025].
Regarding claim 32, GÖRLICH teaches that the VHH antibody is for use in the prevention or treatment of a disorder caused by and/or associated with an infection with SARS-CoV-2 including an infection with a SARS-CoV-2 escape mutant, see para. [0020], [0080].
Regarding claim 33, GÖRLICH teaches that the VHH antibody is for use in the prevention or treatment of Covid-19 in a human subject, see para. [0025], [0081], in particular.
Regarding claims 34-35, GÖRLICH teaches that the VHH antibody is administered orally, nasally, or intrapulmonary, for example by inhalation as an aerosol, see para. [0337], in particular.
Thus, the reference teachings anticipate the claimed invention.
Allowable Subject Matter
An isolated VHH antibody recognizing a SARS-CoV-2 spike protein S1 domain wherein the SARS-CpV-2 spike protein S1 domain is the receptor-binding domain (RBD) of the SARS-CoV-2 S1 domain wherein the VHH comprising a combination of CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 38-40, 18-20, 22-24, 26-28, 30-32, 34-36, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100, 102-104, 106-108, or 110-112. The isolated VHH comprising the amino acid sequence as set forth in in SEQ ID NO: 37, 17, 21, 33, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, or 109 are free of prior art.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-272-0839. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641
