WO2023203135A1 - Anticorps radiomarqué amélioré - Google Patents

Anticorps radiomarqué amélioré Download PDF

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WO2023203135A1
WO2023203135A1 PCT/EP2023/060283 EP2023060283W WO2023203135A1 WO 2023203135 A1 WO2023203135 A1 WO 2023203135A1 EP 2023060283 W EP2023060283 W EP 2023060283W WO 2023203135 A1 WO2023203135 A1 WO 2023203135A1
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antibody fragment
seq
antibody fragments
fragment
population
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PCT/EP2023/060283
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English (en)
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Francis SANTENS
Yana DEKEMPENEER
Sam MASSA
Matthias D'HUYVETTER
Tony Lahoutte
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Precirix N.V.
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Publication of WO2023203135A1 publication Critical patent/WO2023203135A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A61K51/1051—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from breast, e.g. the antibody being herceptin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of methods of preparing populations of labelled antibody fragments.
  • the antibody fragments may be radiolabelled.
  • the labelled antibody fragments are preferably for use in the treatment and/or diagnosis of a cancer.
  • Figure 1 Melting temperature (°C) for anti-HER2 VHH4 with different CARs.
  • Figure 4 In vivo biodistribution of anti-HER2 111 ln-DOTA n -VHH4 with different CARs.
  • Figure 6 Design for ex vivo distribution of anti-FAP 225 ln-DOTA n -VHH1 with different CARs.
  • Figure 7 Ex vivo distribution of anti-FAP 225 ln-DOTA n -VHH1 with different CARs.
  • a method of preparing a population of labelled antibody fragments wherein said population comprises antibody fragments and a number of labels, wherein said antibody fragments comprise a number of conjugation sites which are able to conjugate to said labels, wherein each label is conjugated to one of said conjugation sites
  • the method comprises the steps of: a) providing said antibody fragments; b) providing said labels; c) reacting said labels with said antibody fragments in an aqueous buffer solution, wherein the pH of said aqueous buffer solution is chosen to obtain a given ratio of said number of labels to said number of conjugation sites.
  • the ratio of said number of labels to said number of said conjugation sites is higher than or equal to 0.25 and/or the pH of said aqueous buffer solution is from 9.0 up to 12.0, preferably from 9.0 up to 11.5.
  • a method according to this first aspect may be called a method according to the invention herein.
  • a method according to the invention consists of steps a), b) and c) as defined above.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.50 to X plus 0.50.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.40 to X plus 0.40.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.30 to X plus 0.30.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.25 to X plus 0.25.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.20 to X plus 0.20.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.15 to X plus 0.15.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.10 to X plus 0.10.
  • the pH of the aqueous buffer solution used in a method according to the invention is around 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, 9.75, 10.00, 10.25, 10.50, 10.75, 11.00, 11.25, 11.50, 11.75, or 12.00, wherein around X means from X minus 0.05 to X plus 0.05.
  • aqueous buffers solution is a solution comprising of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it. Hence, they are suitable to maintain the pH chosen in a method according to the invention.
  • the skilled person understands that the pH chosen mandates the use of a suitable buffer, preferably a buffer having a pKa within 1 pH unit of the pH chosen.
  • a buffer solution comprises an acid-base pair having a pKa within 1 pH unit of the pH chosen in a method according to the invention.
  • said acid-base pair is responsible forthe buffering effect of the buffer solution.
  • rLAR relative label-to-antibody fragment ratio
  • a key advantage of a method according to the invention is that the reaction product of step c) can be used for further preclinical, manufacturing or clinical purposes without the need for significant purification.
  • the pH of the aqueous medium is used as the key parameter to obtain the desired population of labelled antibody fragments, eliminating further purification steps which may reduce the yield, increase the time, increase the costs, increase the number of unwanted side products, and/or increase the loss of antibody fragments of the overall reaction.
  • a method according to the invention has an advantage over a corresponding method wherein a purification method is needed to obtain a desired and/or to adjust the rLAR, or a desired standard deviation of the rLAR.
  • These purification methods may result in discarding labelled antibody fragments to obtain a desired rLAR or a desired standard deviation of the rLAR. Such discarding reduces the overall yield of the manufacturing method due to an undesired loss of antibody fragments in the overall reaction.
  • the reaction product of step c) of a method according to the invention comprises a population of labelled antibody fragments having the given ratio of said number of labels to said number of conjugation sites, i.e. the desired rLAR.
  • the population comprised in the reaction product of step c) satisfies any number of other desired characteristics of the population of labelled antibody fragments as described herein.
  • the reaction product of step c) of a method according to the invention of preparing a population of labelled antibody fragments wherein the standard deviation of the rLAR of the population is equal to or lower than 0.25 preferably comprises a population of antibody fragments having a standard deviation of the rLAR equal to or lower than 0.25, without the need for further purification.
  • a method according to the invention comprises the step of d) purifying the reaction product of step c), wherein the product of step c) and d) each comprise a population of labelled antibody fragments, wherein:
  • the absolute difference between the rLAR of these two populations is equal to or lower than 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11 , 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 , preferably 0.05, 0.04, 0.03, 0.02 or 0.01 ; and/or - the absolute difference between the standard deviation of the rLAR of these two populations is equal to or lower than 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11 , 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 , preferably 0.05, 0.04, 0.03, 0.02 or 0.01.
  • the purification step d) does not change rLAR by more than and/or the standard deviation or rLAR by more than 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11 , 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 , preferably 0.05, 0.04, 0.03, 0.02 or 0.01. More preferably, no purification step is comprised in a method according to the invention which does not satisfy at least one of these criteria.
  • a method according to the invention comprises the step of d) formulating the reaction product of step c), wherein step d) comprises a buffer exchange, preferably on an ultrafiltration device.
  • a formulation step preferably does not qualify as a purification step, as described above.
  • a method according to the invention comprises the step of d) purifying the reaction product of step c), wherein the product of step d) can used for further preclinical, manufacturing or clinical purposed without the need for further purification.
  • the pH of the aqueous buffer solution used in a method according to the invention is from 6.0 to
  • the reaction in step c) of a method according to the invention has a reaction time of around 24,
  • X in this context means from 0.95 times X to 1.05 times X.
  • the reaction in step c) of a method according to the invention has a reaction temperature or around 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 0 degrees Celsius.
  • Around X in this context means from 0.95 times X to 1.05 times X.
  • a population of labelled antibody fragments comprising antibody fragments and a number of labels, wherein said antibody fragments comprise a number of conjugation sites which are able to conjugate to said labels, wherein each label is conjugated to one of said conjugation sites, wherein said population is obtainable by a method according to any one of claims 1 to 3, preferably wherein the fraction of antibody fragments conjugated to 0 labels in said population is equal to or lower than 20 percent, preferably for use in the treatment and/or diagnosis of a cancer.
  • a population of antibody fragments according to this second aspect may be called a population of labelled antibody fragments according to the invention or a population according to the invention herein.
  • a population of labelled antibody fragments according to the invention may be characterized by at least one of the following advantages: a higher radiolabelling yield, i.e. if the label is a chelator, the chelation of radionuclides to the labelled antibody fragments comprising the chelators will occur with a higher yield compared to a population of labelled antibody fragments not according to the invention; and/or a higher radiolabelling purity, i.e.
  • the chelation of radionuclides to the labelled antibody fragments comprising the chelators will result in a composition with a higher radiochemical purity, meaning that the ratio of the activity of the radionuclide comprised in the desired radiolabelled antibodies to the total activity of all species containing the radionuclide is higher; and/or a higher stability compared to a population of labelled antibody fragments not according to the invention.
  • a population of labelled antibody fragments according to the invention is more stable than a corresponding population of labelled antibody fragments which does not satisfy the (preferred) rLAR requirements as defined herein.
  • rLAR requirements may be the rLAR, rCAR, LAR, CAR and/or any standard deviation thereof.
  • a higher stability may be expressed as a variety of parameters known to the skilled person, such as higher melting temperature or a lower tendency to aggregate.
  • more stable means a melting temperature which is at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5,
  • more stable means a melting temperature which is from 0.1 ,
  • a lower tendency to aggregate means a 1000-, 100-, 10-, 9-, 8-, 7-, 6-, 5-, 4-, 3-, or 2-fold decrease, wherein the tendency to aggregate may be measured via SE-HPLC.
  • a higher radiolabelling yield means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, or 200% higher.
  • a higher radiolabelling purity means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, or 200% higher.
  • the radiochemical yield and purity are determined by comparing the states right before and right after the chelation of radionuclides, without any purification steps in between.
  • a population of labelled antibody fragments according to the invention is characterized by one or more of the advantages listed above, wherein the binding properties of the labelled antibody fragments (see below) are not or only to some extent adversely affected relative to a corresponding population of labelled antibody fragments not according to the invention.
  • a binding property is k on , k O ff or KD
  • Adversely affected means a higher k O ff, a lower k on and/or a higher KD.
  • to some extent means not more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, 1000%, 2000%, 3000%, 4000%, 5000%, or 10000%.
  • a corresponding population of labelled antibody fragments not according to the invention is preferably a population of labelled antibody fragments that only differs from a similar population according to the invention in LAR, rLAR, or the standard deviation of these properties.
  • any definition, preference or embodiment relating to a labelled antibody fragment of the invention may be applied both to a labelled antibody fragment of the invention and mutatis mutandis to a corresponding method according to the invention.
  • a population of labelled antibody fragments preferably refers to any group of labelled antibody fragments for which statistical mechanical properties can be defined meaningfully.
  • a population preferably refers to at least 10 8 , 10 9 , 10 1 ° or 10 11 labelled antibody fragments.
  • the number of labels and the number of conjugations sites (and their ratios) used herein refer to the total number of labels and conjugation sites comprised in the corresponding population, unless explicitly mentioned otherwise. However, in the population each labelled antibody fragment defines an individual number of conjugation sites, an individual number of labels, and an individual ratio of the individual number of labels to the individual number of conjugation sites. These individual numbers and ratios define a statistical distribution. Wherever a statistic of the population of labelled antibody fragments is mentioned in this application, reference is made to the corresponding statistic of these distributions.
  • the number of labels refers to the total number of labels that are comprised in the population and have been conjugated to a conjugation site. As such, the number of labels should be envisioned as the number of conjugated labels, and does not include any excess of unconjugated labels which could for example be present during various steps in a method according to the invention.
  • the number of conjugation sites is an inherent property of the antibody fragments, meaning that it does not depend on the number of labels. It is defined as the number of sites which are able to conjugate to said labels, but are not necessarily conjugated to one of said labels. The number of conjugation sites is thus the sum of the number of labels (“occupied conjugation sites”) and the number of conjugation sites that are not conjugated to a label (“free conjugation sites”).
  • the ratio of the number of labels to the number of conjugation sites may be called the relative label-to-antibody fragment ratio (rLAR) herein. These terms will be used interchangeable herein.
  • the rLAR can be envisioned as the relative labelling or occupancy of the conjugation sites. Wherever the label comprises a chelator able to coordinate a radionuclide, rLAR may be called the relative chelator-to-antibody fragment ratio (rCAR).
  • the individual rLAR may be defined as the ratio of the individual number of labels to the individual number of conjugation sites comprised in a single antibody fragment comprised in the population of antibody fragments. As explained above, the individual rLAR defines a statistical distribution overthe population of antibody fragments. The rLAR is the average of this distribution.
  • the rLAR and LAR of a population of labelled antibody fragments according to the invention may be measured via an LC-MS method, for example by the one presented in Example 4. Specifically, in embodiments the number of ligands per antibody molecule (LAR) is determined via LC-MS/MS. Based on the sequence of the antibody fragment, the rLAR can then be obtained by dividing the LAR by the number of conjugation sites.
  • the rLAR of a population of labelled antibody fragments according to the invention is equal to or higher than 0.250, 0.255, 0.260, 0.265, 0.270, 0.275, 0.280, 0.285, 0.290, 0.295, 0.300, 0.305, 0.310, 0.315, 0.320, 0.325, 0.330, 0.335, 0.340, 0.345, 0.350, 0.355, 0.360, 0.365, 0.370, 0.375, 0.380, 0.385, 0.390, 0.395, 0.400, 0.405, 0.410, 0.415, 0.420, 0.425, 0.430, 0.435, 0.440, 0.445, 0.450, 0.455, 0.460, 0.465, 0.470, 0.475, 0.480, 0.485, 0.490, 0.495, 0.500, 0.505, 0.510, 0.515, 0.520, 0.525, 0.530, 0.535, 0.540, 0.545, 0.550, 0.555, 0.560,
  • the rLAR of a population of labelled antibody fragments according to the invention is equal to or lower than 1.000, 0.995, 0.990, 0.985, 0.980, 0.975, 0.970, 0.965, 0.960, 0.955, 0.950, 0.945, 0.940, 0.935, 0.930, 0.925, 0.920, 0.915, 0.910, 0.905, 0.900, 0.895, 0.890, 0.885, 0.880, 0.875, 0.870, 0.865, 0.860, 0.855, 0.850,
  • rLAR of the population preferably wherein the standard deviation of the rLAR of the population is equal to or smaller than 0.250.
  • the rLAR of a population of labelled antibody fragments according to the invention is from 0.430 to 0.435, 0.440, 0.445, 0.450, 0.455, 0.460, 0.465, 0.470, 0.475, 0.480, 0.485, 0.490, 0.495, 0.500, 0.505, 0.510, 0.515, 0.520, 0.525, 0.530, 0.535, 0.540, 0.545, 0.550, 0.555, 0.560, 0.565, 0.570, 0.575, 0.580, 0.585, 0.590,
  • the rLAR of a population of labelled antibody fragments according to the invention is from 0.500 to 0.505, 0.510, 0.515, 0.520, 0.525, 0.530, 0.535, 0.540, 0.545, 0.550, 0.555, 0.560, 0.565, 0.570, 0.575, 0.580, 0.585, 0.590, 0.595, 0.600, 0.605, 0.610, 0.615, 0.620, 0.625, 0.630, 0.635, 0.640, 0.645, 0.650, 0.655, 0.660,
  • the rLAR of a population of labelled antibody fragments according to the invention is from 0.250, 0.255, 0.260, 0.265, 0.270, 0.275, 0.280, 0.285, 0.290, 0.295, 0.300, 0.305, 0.310, 0.315, 0.320, 0.325, 0.330,
  • the rLAR of a population of labelled antibody fragments according to the invention is from 0.250,
  • the variance of the rLAR is defined as the expectation ofthe squared deviation of the individual LAR from the mean of the individual LAR, over the population of antibody fragments.
  • the standard deviation of the rLAR is defined as the square root of the variance of the rLAR.
  • the variance and standard deviation of other parameters are defined accordingly herein.
  • Example 1 shows an example of the calculation of the standard deviation of rLAR.
  • the standard deviation of the rLAR of a population of labelled antibody fragments according to the invention is equal to or smaller than 1.00, 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91 , 0.90, 0.89, 0.88, 0.87, 0.86, 0.85, 0.84, 0.83, 0.82, 0.81 , 0.80, 0.79, 0.78, 0.77, 0.76, 0.75, 0.74, 0.73, 0.72, 0.71 , 0.70, 0.69, 0.68, 0.67,
  • the standard deviation of the rLAR of a population of labelled antibody fragments according to the invention is from 0.10 to 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41 , 0.42, 0.43, 0.44, 0.45, 0.46,
  • the standard deviation of the rLAR of a population of labelled antibody fragments according to the invention is from 0.20 to 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31 , 0.32, 0.33, 0.34, 0.35, 0.36,
  • the standard deviation of the rLAR of a population of labelled antibody fragments according to the invention is from 0.50 to 0.51 , 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61 , 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71 , 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81 , 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91 , 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1.00.
  • the ratio of the number of labels to the number of antibody fragments in a population may be called the label-to- antibody fragment ratio (LAR) herein.
  • LAR label-to- antibody fragment ratio
  • the rLAR of the population can be obtained by dividing the LAR by the ratio of the number conjugation sites to the number of antibody fragments.
  • the standard deviation of the LAR is defined analogously.
  • the LAR of a population of labelled antibody fragments according to the invention is equal to or higher than 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0, preferably wherein the standard deviation of the LAR of the population is equal to or smaller than 1.0.
  • the LAR of a population of labelled antibody fragments according to the invention is from 1 .5 to
  • the LAR of a population of labelled antibody fragments according to the invention is from 2.0 to 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6,
  • the fraction of antibody fragments conjugated to 0 or 1 labels in a population according to the invention is equal to or lower than 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15,
  • the fraction of antibody fragments conjugated to 0 labels in a population according to the invention is equal to or lower than 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 , 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1 , 3.0, 2.9,
  • said percentage is defined as a number-weighted average.
  • said percentage may be defined as a mass-weighted average.
  • the relative number of conjugation sites of a population according to the invention is defined as the number of conjugation sites to the number of antibody fragments of the population.
  • the relative number of conjugation sites of a population according to the invention is equal to or higher than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20; or from 2 to 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20; or from 3 to 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20; or from 4 to 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,
  • the standard deviation of the relative number of conjugation sites is preferably lowerthan 0.1 , more preferably lower than 0.01.
  • the standard deviation of the relative number of conjugation sites of a population according to the invention is lower than 0.1 , more preferably lower than 0.01 , most preferably essentially zero.
  • a label comprises a therapeutic or diagnostic group, preferably useful in the treatment or diagnosis of a cancer as discussed herein.
  • the therapeutic or diagnostic group is a radionuclide, more preferably a radionuclide useful in the treatment or diagnosis of a cancer.
  • a label comprises a precursor of a therapeutic or diagnostic group, preferably useful in the treatment or diagnosis of a cancer as discussed herein.
  • a precursor may be for example a prodrug or a chelator that is able to coordinate a radionuclide, wherein said radionuclide is preferably useful in the treatment or diagnosis of a cancer.
  • a label comprises a radionuclide or a chelator able to coordinate a radionuclide.
  • the radionuclide is useful in the treatment or diagnosis of a cancer.
  • a label is a small molecule.
  • a small molecule is defined as an organic molecule or corresponding moiety having a molecular weight of at most 10000 Da, 9000 Da, 8000 Da, 7000 Da, 6000 Da, 5000 Da, 4000 Da, 3000 Da, or 2000 Da, preferably at most 1500 Da, more preferably at most 1000 Da such as at most 900 Da.
  • a label comprises a radionuclide.
  • a population of labelled antibody fragments may be called a population of radiolabeled antibody fragments.
  • a label comprises a radionuclide suitable for therapeutic applications, preferably a radionuclide chosen from the group consisting of a-emitting radioisotopes and p -emitting radioisotopes, including but not limited to a radioisotope chosen from the group consisting of actinium-225, astatine-211 , bismuth-212, bismuth-213, caesium-137, chromium-51 , cobalt-60, copper-67, dysprosium-165, erbium-169, fermium-255, gold-198, holium- 166, iodine-125, iodine-131 , iridium-192, iron-59, lead-212, lutetium-177, molybdenum-99, palladium-103, phosphorus-32, potassium-42, rhenium-186, rhenium-188, samarium-153, radium-223 , radium-
  • a label comprises iodine-131.
  • a label comprises actinium-225, bismuth-213, iodine-125, iodine-131 , lutetium-177, yttrium-90, copper-67, rhenium-186, rhenium-188, terbium-149, or terbium-161.
  • a label comprises actinium-225 or bismuth-213, most preferably actinium-225.
  • a label comprises lutetium-177 or terbium-161 , most preferably lutetium-177.
  • a label comprises a radionuclide suitable for diagnostic applications, preferably a radionuclide chosen from the group consisting of positron-emitting radioisotopes (PET) or y-emitting radioisotopes (SPECT), including but not limited to a radioisotope chosen from the group consisting of iodine-131 , yttrium-90, iodine-125, lutetium-177, rhenium-186, rhenium-188, scandium-43, scandium-44, technetium-99m, terbium-161 , terbium-149, indium-111 , xenon-133, thallium-201 , fluorine-18, gallium-68, gallium-67, copper-67, iodine-123, iodine-124, zirconium-89 and copper-64.
  • a label comprises iodine-131 .
  • a label comprises actinium-225 or bismuth-213, most preferably said label further comprises a chelator able to coordinate these radionuclides such as DOTA.
  • a label comprises lutetium-177 or terbium-161 , most preferably said label further comprises a label able to coordinate these radionuclides such as DOTA.
  • a label comprises indium-111 , actinium-225 or gallium-68, preferably said label comprises indium- 111 , actinium-225 or gallium-68, and a chelator able to bind these radionuclides such as DOTA.
  • a label comprises indium-111 or actinium-225, preferably said label comprises indium-111 , actinium-225, and a chelator able to bind these radionuclides such as DOTA.
  • a label comprises a chelator.
  • a chelator as defined herein is able to coordinate a radionuclide, typically via is a non-covalent, coordinative bond. Such a chelator may or may not be coordinated to a radionuclide.
  • a label may comprise both a chelator able to coordinate a radionuclide and a radionuclide.
  • rLAR may be called rCAR.
  • a method according to the invention comprises the step of e) contacting the product of step c) or of step d) with radionuclides as described herein.
  • a chelator comprises a macrocycle.
  • Preferred chelators comprising a macrocycle include, but are not limited to, 1 ,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA), 1 ,4,7, 10,13,16- hexaazacyclohexadecane-N,N',N",N"',N"",N"'"-hexaacetic acid (HEHA), 1 ,4,7,10,13- pentaazacyclopentanadecane- N,N',N",N"', N""-pentaacetic acid (PEPA), Macropa (Thiele et al., An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy.
  • DOTA 1 ,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraace
  • a chelator is 1 ,4,7, 10-tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (DOTA), 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA), Macropa, PCTA or Crown.
  • a chelator is 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10- tetraacetic acid (DOTA) or 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA).
  • a chelator comprises an open chain ligand.
  • Preferred chelators comprising an open chain ligand are not limited to, deferoxamine (DFO), ethylenediaminetetraacetic acid (EDTA), and diethylenetriaminepentaacetic acid (DTPA).
  • DFO deferoxamine
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • a label comprises an iodine isotopes and a benzoate linker, preferably 3-pyridinecarboxyl, benzoate or guadinomethyl benzoate, even more preferably guadinomethyl benzoate, most preferably 4- guadinomethyl benzoate.
  • a benzoate linker preferably 3-pyridinecarboxyl, benzoate or guadinomethyl benzoate, even more preferably guadinomethyl benzoate, most preferably 4- guadinomethyl benzoate.
  • a label is a non-radioactive label.
  • such non-radioactive label is a fluorescent label.
  • a label may be used for diagnostic applications as defined herein. Alternative applications include image-guided surgery or photodynamic therapy. Examples of suitable fluorescent labels for diagnostic applications include Alexa fluor variants, Cy3, Cy5, FITC (fluorescein), Coumarin, Texas red, Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, PE-Cyanine7, PerCP-Cyanine5.5, TRITC (tetramethylrhodamine).
  • fluorescent labels for image-guided surgery examples include IRDye800CW, IRDye680-RD, ZW800-1 , FNIR (see for example Pieterjan Debie et al, Front Pharmacology, 2019; 10:510, doi: 10.3389/fphar.2019.00510, PMCID: PMC6527780, PMID: 31139085).
  • Example of a suitable fluorescent label for photodynamic therapy includes IRDye700DX. Most labels may be obtained from ThermoFisher or from Licor.
  • a label is a molecule to be delivered to a cell, a tissue, an organ expressing human and/or murine FAP. Any moiety, molecule or medicament known to act on a cell, tissue, organ expressing FAP is potentially encompassed by these embodiments.
  • the molecule may be a peptide, a small molecule or a nucleic acid.
  • a peptide may be a cytokine.
  • a small molecule may be a chemotherapeutic.
  • a label is an AcTakine (Activity-on-Target cytokine) or an AcTaferon (IFNa-based AcTakine), preferably an AcTakine or an AcTaferon as described in WO2017077382A1 , W02017134301A1 , WO2017194783A1 , WO2017194782A2, WO2018077893A1 , WO2018141964A1 , WO2018144999A1 ,
  • the label is preferably a medicament for cancer.
  • a label is a pyrrolobenzodiazepine; preferably a pyrrolobenzodiazepine dimer such as described in W02014057074A1 , WO2015052322A1 , W02014140174A1 , WO2015052321 A1 , WO2017186894A1 , WO2017137555A1 , WO2017137553A1 , WO2016038383A1 or WO2018192944A1 ; more preferably herein said pyrrolobenzodiazepine dimer is selected from the group consisting of:
  • the label is preferably a medicament for cancer.
  • a label is an octadentate thorium chelator such as described in W02017211809A1.
  • the label is preferably a medicament for cancer.
  • a label is a dolastatin or an auristatin as described in WO2015162293A1.
  • the label is preferably a medicament for cancer.
  • a label is cytolysin or a Nigrin-b A-chain such as described in WO2015118030A2.
  • the label is preferably a medicament for cancer.
  • a label is 2-propylthiazolo [4, 5-c] quinolin-4-amine, 1-(2-methylpropyl)-1 H-imidazo[4,5-c]quinolin- 4-amine, 4-amino-2-(ethoxymethyl)-a, a-di-methyl-1 H-imidazo[4,5-c]quinoline-1 -ethanol, 1-(4-amino-2- ethylaminomethylimidazo-[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol,N-[4-(4-amino-2-ethyl-1 H-imidazo[4,5- c]quinolin-1-yl)butyl-]methanesulfonamide,4-amino-2-ethoxymethyl-aa-dimethyl-6,7,8,9-tetrahydro-1 h- imidazo[4,5-c]quinoline-1 -ethanol, 4-amino-aa-dimethyl-2-methoxye
  • a label is a Pseudomonas exotoxin such as described in W02015051199A2.
  • the label is preferably a medicament for cancer.
  • WO2019191519A1 and W02020033646A1 are incorporated in their entirety, and all compounds disclosed therein may be a label in the context of the current application.
  • a label is a prodrug of adrenomedullin as described in W02013064508A1 , an autotaxin inhibitor as described in WO2014097151A2, a pyrimido[4,5-b]quinoline-4,5 (3h,10h)-dione derivative as described in WO2014091446A1 , an amiloride derivative as described in W02013064450A1 , a pyrrolo[2,3-d]pyrimidine derivative as described in WO2014177527A1 , a pyrazolopyridine derivative or a pyrazolopyrimidine derivative as described in WO2015173683A1 , a piperidino-dihydrothienopyrimidine sulfoxide derivative as described in WO201326797A1 , a 2-[pyridin-3-yl]-2,3-dihydro-benzo[1 ,4]dioxine derivative as described in W02016061161A1 , a
  • WO201760879A1 W02018109607A1 , WO201728926A1 , WO201287519A1 , WO201671375A1 ,
  • WO2015104354A1 , W0201415905A1 and W02005104745A1 are incorporated in their entirety, and all compounds disclosed therein may be a label in the context of the current application.
  • a label is a molecule to be delivered to a cell, a tissue, an organ expressing human FOLR1. Any moiety, molecule or medicament known to act on a cell, tissue, organ expressing FOLR1 is potentially encompassed by these embodiments.
  • a label is a molecule to be delivered to the central nervous system (CNS). More preferably, the molecule is a medicament acting in the CNS, preferably acting in the brain. Even more preferably, the molecule crosses the brain blood barrier (BBB) and/or the blood-cerebrospinal fluid barrier (BCSFB) via transport via the human FOLR1 ; a mechanism called receptor mediated transcytosis (RMT).
  • BBB brain blood barrier
  • BCSFB blood-cerebrospinal fluid barrier
  • RMT receptor mediated transcytosis
  • Any moiety, molecule or medicament known to act in the CNS or in the brain is potentially a label in the context of the present invention.
  • Such a moiety, molecule or may be any molecule or medicament, which does not cross the brain blood barrier (BBB) on its own.
  • the molecule may be a peptide, a small molecule or a nucleic acid.
  • a peptide may be a cytokine.
  • a small molecule
  • a medicament acting in the brain is a medicament for preventing and/or treating choroid plexus papilloma and/or hydrocephalus.
  • Folate receptor alpha (FOLR1 ) is overexpressed in some human cancers, including choroid plexus papilloma ortumour.
  • An increased expression of folate receptor alpha (FOLR1 ) in the brain has been associated with hydrocephalus.
  • a label is an aziridinyl-epothilone such as described in W02007140297A2, preferably a label is 7, 11-dihydroxy-17-[2- hydroxyethyl]-8,8,10,12-tetramethyl-3-[1-methyl-2-(2-methyl- 4- thiazolyl)ethenyl]-4-oxa-17- azabicyclo[14.1.0]heptadecane-5, 9-dione, or a derivative thereof, or a pharmaceutically acceptable salt thereof, more preferably said label is a compound for the treatment and/or prevention of choroid plexus papilloma, most preferably said label is comprised in a population according to the invention for the treatment and/or prevention of choroid plexus papilloma.
  • a label is a 1 H-pyrrolo[3,2-b]pyridine derivative such as described in W02014145051A1 , preferably a label is 4-(6-(3,5-dimethylisoxazol-4-yl)-1-(1-(pyridin-2-yl)ethyl)-1 H-pyrrolo[3,2-b]pyridin-3-yl)benzoic acid, or 4-(6-(3,5-dimethylisoxazol-4-yl)-1-(phenyl(pyridin-2-yl)methyl)-1 H-pyrrolo[3,2-b]pyridin-3-yl)benzoic acid, or methyl 2-(4-(6-(3,5-dimethylisoxazol-4-yl)-1-(1-(pyridin-2-yl)ethyl)-1 H-pyrrolo[3,2-b]pyridin-3-yl)phenyl)acetate, or methyl 4-(6-(3,5-dimethylis
  • a label is a 1 H-pyrrolo[3,2-b]pyridine derivative such as described in WO2017053243A1 , preferably a label is 4-( 1 -( 1 , 1 -di(pyridin-2-yl)ethyl)-6-(3,5-dimethylisoxazol-4-yl)-1 H-pyrrolo[3,2-b]pyridin-3- yl)benzoic acid, or 4-(1-(cyanodipyridin-2-ylmethyl)-6-(3,5-dimethylisoxazol-4-yl)-1 H-pyrrolo[3,2-b]pyridin-3- yl)benzoic acid, or 4-(6-(3,5-dimethylisoxazol-4-yl)-1-(fluorodipyridin-2-ylmethyl)-1 H-py rrolo[3,2-b]py rid in-3- yl)benzoic acid, or a derivative thereof, or a pharmaceutical
  • a label is an pyrrolidine sulfonamide TRPC4 antagonists such as described in WO2018055524A1 , preferably a label is 2-(((3R,4S)-4-(4-chlorophenoxy)-3-hydroxy-3-(hydroxymethyl)pyrrolidin-1-yl)sulfonyl)-5- (trifluoromethyl)benzonitrile, or 4-(((3S,4S)-1-((2-cyano-4-(trifluoromethyl)phenyl)sulfonyl)-4-hydroxy-4-((S)-1- hydroxyethyl)pyrrolidin-3-yl)oxy)-2-fluorobenzonitrile, or a derivative thereof, or a pharmaceutically acceptable salt thereof, more preferably said label is a compound for the treatment and/or prevention of hydrocephalus, most preferably said label is comprised in a population of antibody fragments according to the invention for the treatment and/or prevention of hydrocephalus.
  • a label is a sulfonpyrazole or sulfonylpyrazoline carboxamidine 5-HT6 antagonist such as described in W02008034863A2, preferably a label is N'-(1 -acetylindolin-5-ylsulfonyl)-N,4-diethyl-4,5-dihydro-1 H- pyrazole-1-carboximidamide, or N'-(4-(1 H-pyrazol-1-yl)phenylsulfonyl)-N,4-diethyl-4,5-dihydro-1 H-pyrazole-1- carboximidamide, or N-((5-(N-((4-ethyl-4,5-dihydro-1 H-pyrazol-1-yl)(ethylamino)methylene)sulfamoyl)thiophen-2- l)methyl)benzamide, or N'-(5-(5-(chloromethyl)-1 ,2,
  • a label is an arylsulfonyl pyrazoline carboxamidine 5-HT6 antagonist such as described in W02009115515A1 , preferably a label is N'-(4-amino-3-chlorophenylsulfonyl)-N-ethyl-2,3,8-triazaspiro[4.5]dec-3- ene-2-carboximidamide, or N'-(4-amino-3-chlorophenylsulfonyl)-N-ethyl-2,3-diazaspiro[4.4]non-3-ene-2- carboximidamide, or N'-(4-amino-3-chlorophenylsulfonyl)-N-ethyl-4,4-dimethyl-4,5-dihydro-1 H-pyrazole-1- carboximidamide , or N'-(4-aminophenylsulfonyl)-2,3-diazas
  • a label is a cyclodextrin-API conjugate such as described in WO2013116200A1 , more preferably said label is a compound for the treatment and/or prevention of hydrocephalus, most preferably said label is comprised in a population of antibody fragments according to the invention for the treatment and/or prevention of hydrocephalus.
  • a label is pyrrolidine sulfonamide TRPV4 antagonists such as described in WO2018055527A1 , preferably a label is 4-(((3S,4R)-1-((2,4-dichlorophenyl)sulfonyl)-4-hydroxy-4-(hydroxymethyl)pyrrolidin-3- yl)methyl)-2-fluorobenzonitrile, or 4-(((3S,4R)-1-((2-chloro-4-(trifluoromethyl)phenyl)sulfonyl)-4-hydroxy-4- (hydroxymethyl)pyrrolidin-3-yl)methyl)benzonitrile, or 4-(((3S,4R)-1-((5-chloropyridin-2-yl)sulfonyl)-4-hydroxy-4- (hydroxymethyl)pyrrolidin-3- yl)methyl)-3-(2,2,2-trifluoroethoxy)benzonitrile, or 4-(((3S,4R)-1
  • a label is [5-(5-chloro-1 H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-pyridin-2-yl]- (6-trifluoromethyl-pyridin- 3-ylmethyl)-amine such as described in WO2016179415A1 , or a derivative thereof, or a pharmaceutically acceptable salt thereof, preferably said label is a compound for the treatment and/or prevention of hydrocephalus, most preferably said label is comprised in a population of antibody fragments according to the invention for the treatment and/or prevention of hydrocephalus.
  • a label is a N-(pyridin-3-ylmethyl) substituted 1 H-pyrrolo[2,3-b]pyridine derivative such as described in WO2013142427A1 or a 1 H-pyrrolo[2,3-b]pyridine derivatives such as described in WO2017100201 A1 , or a derivative thereof, or a pharmaceutically acceptable salt thereof, preferably said label is a compound for the treatment and/or prevention of hydrocephalus, most preferably said label is comprised in a population of antibody fragments according to the invention for the treatment and/or prevention of hydrocephalus.
  • W02009115515A1 , W02013116200A1 , WO2018055527A1 , WO2016179415A1 , WO2013142427A1 , and W02017100201A1 are incorporated in their entirety, and all compounds disclosed therein may be a label in the context of the current application. Conjugation sites and chemistry
  • a conjugation site comprises a primary amine.
  • the number of said conjugation sites is preferably equal to the number of primary amines comprised in said antibody fragments.
  • These primary amines may be the amino terminus or any lysine residue comprised in the antibody fragments, but could also be any primary amines comprised in non-natural or modified amino acids residues.
  • a conjugation site is a lysine residue or an amino terminus of an antibody fragment comprised in a population according to the invention.
  • the number of said conjugation sites is equal to the number of lysine residues comprised in said antibody fragments plus the number of amino-termini comprised in said antibody fragments.
  • a label as described herein is conjugated to a conjugation site. This conjugation is established during the reaction in step c) of a method according to the invention.
  • c) reacting said labels with said antibody fragments comprises a reaction between an isothiocyanate, a N-hydroxysuccinimide (NHS), an acyl fluoride, a sulfonyl fluoride, an imminoboronate, a diazonium salt or an aldehyde, preferably an isothiocyanate; and a primary amine comprised in said conjugation sites, preferably wherein said conjugations sites are independently lysine residues or amino termini.
  • NHS N-hydroxysuccinimide
  • the labels provided in step b) comprise an isothiocyanate group, a N- hydroxysuccinimide (NHS), an acyl fluoride, a sulfonyl fluoride, an imminoboronate, a diazonium salt or an aldehyde, preferably an isothiocyanate.
  • N- hydroxysuccinimide NHS
  • an acyl fluoride a sulfonyl fluoride
  • an imminoboronate a diazonium salt or an aldehyde
  • aldehyde preferably an isothiocyanate.
  • the term “antibody fragment” refers to any fragment of an antibody or immunoglobulin.
  • the antibody fragment is a single-domain antibody fragment.
  • the antibody fragment is a heavy chain variable domain derived from a heavy chain antibody (VHH) or a fragment thereof.
  • a single-domain antibody fragment is a VHH or a fragment thereof: the heavy chain variable domains derived from heavy chain antibodies (i.e. the VHH’s) as disclosed herein consist of a single polypeptide chain.
  • antibody fragment may be replaced by “single-domain antibody fragment” or by “VHH” or by “a fragment of a VHH” or by “a functional fragment of a VHH”.
  • a fragment of an antibody or of a VHH is a functional fragment as it exhibits at least an activity of the antibody or of the VHH to some extent. “Some extent” may mean at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more.
  • a preferred activity of the antibody fragment, VHH or a fragment of a VHH is the specific binding to human and/or murine FAP, human FOLR1 and/or human HER2.
  • the heavy chain variable domains derived from heavy chain antibodies (VHH’s), or fragments thereof, as disclosed herein consist of a single polypeptide chain. More particularly, the VHH’s are derived from an innate or adaptive immune system, preferably from a protein of an innate or adaptive immune system. Still more particularly, the VHH’s disclosed herein may comprise 4 framework regions (FR) and 3 complementary determining regions (CDR), or any suitable fragment thereof (which will then usually contain at least some of the amino acid residues that form at least one of the CDR). In particular, the VHH’s disclosed herein are easy to produce at high yield, preferably in a microbial recombinant expression system, and convenient to isolate and/or purify subsequently.
  • FR framework regions
  • CDR complementary determining regions
  • the VHH’s as disclosed herein may comprise CDR (complementarity determining regions) sequences of antibodies (or may be based on and/or derived from such CDR sequences, as further described herein), they will also generally be referred to herein as ‘CDR sequences’ (i.e. as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively).
  • CDR sequences i.e. as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively.
  • the VHH’s as disclosed herein comprise at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein.
  • VHH’s as disclosed herein have the (general) structure:
  • IMGT nomenclature is used to define the FR (framework regions) FR1 , FR2, FR3 and FR4 and corresponding CDR regions CDR1 , CDR2, and CDR3.
  • FR framework regions
  • CDR1 , CDR2, and CDR3 corresponding CDR regions
  • a fragment of a VHH preferably exhibits at least an activity of the VHH to some extent. “Some extent” may mean at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more.
  • a preferred activity of a fragment of a VHH is the specific binding to an antigen, preferably with a dissociation constant (k O ff) equal to or smallerthan 10' 1 s' 1 , preferably equal to or smallerthan 10' 2 s' 1 , more preferably equal to or smallerthan 10' 3 s' 1 , most preferably equal to or smaller than 10' 4 s' 1 .
  • k O ff dissociation constant
  • a more preferred activity of a fragment of a VHH is the specific binding to an antigen with a dissociation constant (k O ff) in the range from 10' 1 s' 1 to 10' 5 s' 1 , preferably in the range from 10' 2 s' 1 to 10' 5 s' 1 , more preferably in the range from 10' 3 s' 1 to 10' 5 s' 1 , most preferably in the range from 10' 4 s' 1 to 10' 5 s' 1 .
  • k O ff dissociation constant
  • a fragment of a VHH preferably has a length from 20 to 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids, or from 50 to 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 140 or 150 amino acids.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind an antigen, wherein said antigen is comprised in a target, wherein said binding of said antibody fragment to said antigen does cause a significant conformational change in said target.
  • an antibody fragment is able to specifically bind an antigen associated with and/or overexpressed in a tumor and/or present on the surface of a cancer cell.
  • said antigen is CEA, PSMA, 4lg-B7-H3, A33, a5li1 Integrin, AGS-16, AGS-8, avli3 Integrin, CAIX/MN, CCR-2/CCL-2, CD40, CD44v6, CK19, CTLA-4, d9-E-cadherin, DNA/histone H1 , ED-B, fibronectin, EFGL7, EGFR, EGP-1 , Endoglin, EpCAM, FAP, FZD10, GC128, GD2, GD3, HER-2, HER-3, HPV-16 E6, IGF-1 R, IL-6, L1-CAM, Lewis y , MAGE-A3, Melanin, Mesothelin, MET/HGF, MUC1 , NCAM, Neuropilin-1 , PD-1/PD-L1 , PDGFR, PSCA, TAG-72, TEM-1 , Tenascin-C,
  • the methods and populations according to the invention are not limited to a specific antigen associated with and/or overexpressed in a cancer. Without being bound to this theory, the pH-mediated rLAR tuning, and any associated with it, may be translated to other antigens than the ones explicitly described below.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind an antigen with a dissociation constant (k O ff) equal to or smaller than 10' 1 s' 1 , preferably equal to or smaller than 10' 2 s' 1 , more preferably equal to or smaller than 10' 3 s' 1 , most preferably equal to or smaller than 10' 4 s' 1 .
  • k O ff dissociation constant
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind an antigen with a dissociation constant (k O ff) in the range from 10' 1 s' 1 to 10' 5 s' 1 , preferably in the range from 10' 2 s' 1 to 10' 5 s' 1 , more preferably in the range from 10' 3 s' 1 to 10' 5 s' 1 , most preferably in the range from 10' 4 s' 1 to 10' 5 s' 1 .
  • k O ff dissociation constant
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind an antigen, wherein said antigen is present on the surface of a tumour cell or in a tumour microenvironment, more preferably with a dissociation constant (k O ff) equal to or smaller than 10' 1 s' 1 , preferably equal to or smaller than 10' 2 s' 1 , more preferably equal to or smaller than 10' 3 s' 1 , most preferably equal to or smaller than 10' 4 s' 1 .
  • k O ff dissociation constant
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind an antigen, wherein said antigen is present on the surface of a tumour cell or in a tumour microenvironment, more preferably with a dissociation constant (k O ff) in the range from 10' 1 s' 1 to 10' 5 s' 1 , preferably in the range from 10' 2 s' 1 to 10' 5 s' 1 , more preferably in the range from 10' 3 s' 1 to 10' 5 s' 1 , most preferably in the range from 10' 4 s' 1 to 10' 5 s' 1 .
  • k O ff dissociation constant
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind an antigen, wherein said antigen is an oncogene and/or wherein said antigen is expressed on a cancer cell or in a tumour microenvironment, more preferably with a dissociation constant (k O ff) equal to or smaller than 10' 1 s' 1 , preferably equal to or smaller than 10' 2 s' 1 , more preferably equal to or smaller than 10' 3 s' 1 , most preferably equal to or smaller than 10' 4 s' 1 .
  • k O ff dissociation constant
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind an antigen, wherein said antigen is an oncogene and/or wherein said antigen is expressed on a cancer cell or in a tumour microenvironment, more preferably with a dissociation constant (k O ff) in the range from 10' 1 s' 1 to 10' 5 s' 1 , preferably in the range from 10' 2 s' 1 to 10' 5 s' 1 , more preferably in the range from 10' 3 s' 1 to 10' 5 s' 1 , most preferably in the range from 10' 4 s' 1 to 10' 5 s' 1 .
  • k O ff dissociation constant
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is able to specifically bind human fibroblast activation protein (FAP), human folate receptor alpha (FOLR1 ) or human epidermal growth factor receptor 2 (HER2), preferably human fibroblast activation protein (FAP) or human folate receptor alpha (FOLR1 ).
  • FAP human fibroblast activation protein
  • FOLR1 human folate receptor alpha
  • HER2 human epidermal growth factor receptor 2
  • FAP human fibroblast activation protein
  • FOLR1 human folate receptor alpha
  • HER2 human epidermal growth factor receptor 2
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is particularly suited for binding to human and/or murine FAP.
  • the antibody fragment, or the VHH or fragment thereof specifically binds human and murine FAP.
  • the antibody fragment, or the VHH or fragment thereof binds part of the extracellular domain of human and/or murine FAP.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is preferably used for treating a cancer associated with the expression of human FAP in a CAF cell and/or in a cancer cell.
  • the label may be a medicament to treat fibrosis, wound healing, myocardial infarction, atherosclerosis, arthritis and other inflammatory and fibrotic diseases.
  • the labelled antibody fragment is used to target the medicament to the site of the disease listed in order to treat it.
  • Human FAP is quite attractive to be targeted as it is specifically expressed and more specifically overexpressed in cancer-associated fibroblasts (CAF) which have a tumourigenic function (Pure et al 2018, Oncogene Aug;37(32):4343-4357). It is also expressed in some cancer cells (such as leukemia, bone, uterus, pancreas, skin, muscle, brain, breast, colorectal, oesophageal, gastric, liver, lung, ovarian, parathyroid, renal cancer as disclosed later herein) and poorly expressed in healthy cells. Human FAP may therefore be considered as a tumour antigen or a cancer cell antigen and may therefore be used as diagnostic and/or therapeutic target.
  • CAF cancer-associated fibroblasts
  • the antibody fragment, or the VHH or fragment thereof are also encompassed by the present invention.
  • Such other diagnostic and/or therapeutic applications are not linked to cancer but may be linked to fibrosis, wound healing, myocardial infarction, atherosclerosis, arthritis and other inflammatory and fibrotic diseases.
  • the antibody fragment, or the VHH or fragment thereof may be used in a diagnostic and/or therapeutic application to diagnose and/or treat fibrosis, wound healing, myocardial infarction, atherosclerosis, arthritis and other inflammatory and fibrotic diseases. More detailed explanation is given later herein.
  • human FAP is considered a ‘cancer cell-specific antigen’, ‘cancer-specific antigen’, ‘cancer antigen’, ‘target protein present on, 'target protein expressed in' and/or 'specific for a cancer cell’, ‘cancer cell-specific target (protein)', 'cancer (cell)-associated antigen' are used interchangeably herein and refers to the fact that human FAP is mainly present on (or mainly expressed on) cancer cells and in the vicinity of a tumour and/or in the vicinity of metastases. FAP is specifically expressed and more specifically overexpressed in cancer-associated fibroblasts (CAF) which have a tumourigenic function.
  • CAF cancer-associated fibroblasts
  • FAP is poorly expressed in healthy cells.
  • Human FAP may therefore be considered as a tumour antigen or a cancer cell antigen and may therefore be used as diagnostic and/or therapeutic target.
  • human FAP is expressed in cancer- associated fibroblast.
  • FAP positive or “expressing FAP” or “overexpressing FAP” may refer to cancerous or malignant human cells and/or cancer-associated fibroblasts or tissue characterized by FAP protein overexpression and thus have abnormally high levels of the FAP gene and/or the FAP protein compared to normal healthy cells.
  • overexpressing may mean that the expression is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than the expression in a control cell line.
  • a control cell line may be a healthy or non-diseased cell.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof, specifically binds human and/or murine FAP, and fulfils at least one of the following: a. the epitope is comprised within amino acid 26 to 760 of SEQ ID NO: 5, preferably the epitope is comprised within (or comprises) amino acids 65-90 and/or 101-140 of SEQ ID NO: 5, b. wherein at least amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83,
  • said antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 80% sequence identity with at least one of SEQ ID NO: 1 , 2, 3, 4 or a portion thereof.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof, specifically binds human and/or murine FAP, and fulfils at least one of the following: a. the epitope is comprised within amino acids 26 to 760 of SEQ ID NO: 5, preferably the epitope is comprised within (or comprises) amino acids 65-90 and/or 101-140 of SEQ ID NO: 5, b. the antibody fragment, or the VHH or fragment thereof, specifically binds to the following amino acids of SEQ ID NO: 5:
  • the antibody fragment, or the VHH or fragment thereof is represented by an amino acid sequence that comprises an amino acid sequence having at least 80% sequence identity with at least one of SEQ ID NO: 1 , 2, 3, 4 or a portion thereof.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds human and/or murine FAP, and has its epitope comprised within amino acids 26 to 760 of SEQ ID NO: 5, specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds human and/or murine FAP, and has its epitope comprised within (or has its epitope which comprises) amino acids 65-90 and/or 101-140 of SEQ ID NO: 5, specifically binds to the following amino acids of SEQ ID NO: 5:
  • FAP is synonymous with FAPalpha and corresponds to the polypeptide Prolyl endopeptidase FAP, which is also named Fibroblast activation protein alpha (FAPalpha).
  • FAPalpha Fibroblast activation protein alpha
  • the binding to human and/or murine FAP may be assessed in a homogeneous mixture of different antigens.
  • a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000- fold).
  • the binding may be assessed in vitro using cells expressing human and/or murine FAP, and optionally in vivo or ex vivo as earlier defined herein.
  • These cells may be human cells and expressing endogenous human and/or murine FAP. Alternatively, these cells may overexpress human and/or murine FAP.
  • Cells overexpressing human and/or murine FAP may be human or non-human cells.
  • Preferred cells are fibroblast cells expressing human and/or murine FAP.
  • a preferred transfected cell line is HEK293.
  • a preferred cell line expressing FAP is GM05389 or U-87 MG.
  • a preferred cancer cell line expressing human FAP is U-87 MG.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to human and/or murine FAP.
  • This assessment is preferably carried out using ELISA, Surface Plasmon Resonance or Bio-Layer Interferometry.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof will bind to a number of naturally occurring or synthetic analogues, variants, mutants, alleles, parts and fragments of human and/or murine FAP.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof will specifically bind to at least those analogues, variants, mutants, alleles, naturally occurring, synthetic analogues, parts and fragments of human and/or murine FAP that (still) contain the epitope of the (natural/wild-type) antigen to which the antibody fragment binds.
  • the epitope of human FAP of an antibody fragment is comprised within amino acids 26 to 760 of SEQ ID NO: 5.
  • the epitope of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is comprised within amino acids 65-90 and/or 101-140 of SEQ ID NO: 5.
  • the epitope of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprises the amino acid stretch or region 65-90 and/or 101-140 of SEQ ID NO: 5.
  • the epitope of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprises the combination of amino acid stretch or region 65-90 and 101-140 of SEQ ID NO: 5.
  • the epitope of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is comprised within the combination of amino acid stretch or region 65-90 and 101-140 of SEQ ID NO: 5.
  • At least one of the following amino acids of SEQ ID NO: 5 is bound or contacted or interacts with an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91 , L105, S106, P107, D108, R109, Q110, F111 , D134, L135, S136, N137, V158, G159, R175, D457 and Y458.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 5:
  • amino acids of SEQ ID NO: 5 are bound or contacted or interacted with an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91 , L105, S106, P107, D108, R109, Q110, F111 , D134, L135, S136, N137, V158, G159, R175, D457 and Y458.
  • an amino acid of human of FAP may be bound by the antibody fragment, or the VHH or fragment thereof, when said amino acid belongs to the epitope of the antibody fragment.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is said to be 'specific' for a first target antigen of interest (i.e. human and/or murine FAP) as opposed to a second molecule, such as one of the closest homologues of FAP (i.e.
  • DPP IV Dipeptidyl amino-peptidase IV, Juillerat-Jeanneret, L.,et al (2017), Expert Opinion on therapeutic targets, 21 : 977-991 ) when it binds to the first target antigen of interest with an affinity that is at least 5 times, such as at least 10 times, such as at least 100 times, and preferably at least 1000 times higher than the affinity with which that the antibody fragment binds to the second molecule.
  • the amino acid sequence of DPP IV has 52% identity with the amino acid sequence of FAP and still the antibody fragment, or the VHH or fragment thereof, can distinguish between the two related prolyl-specific serine proteases.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is said to be ‘specific for’ a first target antigen of interest as opposed to a second molecule, it may specifically bind to (as defined herein) the first target antigen of interest, but not to the second molecule.
  • a preferred cell used for testing the in vitro binding to human and/or murine FAP is a cell expressing human and/or murine FAP.
  • a preferred cell line may be GM05389, U-87 MG or transfected HEK293 as defined earlier herein.
  • ‘competing (with)’, 'cross-blocking', 'cross-binding' and 'cross-inhibiting' using an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may mean interfering with or competing with the binding of another antibody fragment as described herein with human and/or murine FAP, thereby reducing that binding by at least 10% but preferably at least 20%, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more, as measured using a suitable in vitro, cellular or in vivo assay, compared to the binding of that other antibody fragment as described herein with human and/or murine FAP but without using the 'cross-blocking' antibody fragment.
  • an antibody fragment does not compete with the ligand of FAP for binding to it.
  • the antibody fragment, or the VHH or fragment thereof is also expected not to interfere with the natural function of this receptor. It means that in an embodiment, an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof does not compete with the natural ligand of human and/or murine FAP and therefore is not inhibited to bind to human and/or murine FAP-expressing cells in vitro or in an in vivo or ex vivo setting.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof does not substantially alter a FAP activity, it means it preferably does not substantially inhibit a FAP activity.
  • 'substantial' may mean at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of a FAP activity is still detectable compared to the same FAP activity when the antibody fragment, or the VHH or fragment thereof, is not present.
  • a FAP activity may be an exopeptidase and/or an endopeptidase activity.
  • This exopeptidase activity may be a FAP dipeptidyl peptidase activity.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof does not substantially inhibit the FAP dipeptidyl peptidase activity.
  • This endopeptidase activity may be a gelatinase and/or collagenase activity.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof does not substantially inhibit the FAP gelatinase and/or collagenase activity of FAP.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is not a modulator of human and/or murine FAP. In an embodiment, it is not an inhibitor and it is not an activator of human and/or murine FAP. Any of the FAP activities may be assessed as illustrated in Juillerat-Jeanneret L. et al, (2017), Expert Opinion on Therapeutic Targets (http://dx.doi.org/10.1080/14728222.2017.1370455). The FAP dipeptidyl peptidase activity may be assessed using techniques known to the skilled person. In short, the human FAP enzymatic activity may be measured using the fluorogenic substrate benzyloxycarbonyl-Gly-Pro-7-amido-4- methylcoumarin (Z-Gly-Pro-AMC; Bachem).
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is said to show 'cross-reactivity' for two different target proteins of interest if it is specific for (as defined herein) both of these different target proteins of interest.
  • the two different target proteins of interest may be human and murine FAP.
  • first, second, third, fourth structural features of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof.
  • the antibody fragment, or the VHH or fragment thereof may be characterized by the presence of at least one, or all of these four structural features: third and fourth structural features, first and second structural features, first, second and third structural features third and fourth structural features, third structural feature, fourth structural feature, first, third and fourth structural features, Second, third and fourth structural features, first and third structural features, first and fourth structural features, second and third structural features, second and fourth structural features first, second, third and fourth structural features.
  • First structural feature of an antibody fragment based on the contacted region of FAP.
  • a first structural feature is that the antibody fragment contacts or binds or specifically binds or interacts to a region of human FAP comprised within amino acid 26 to 760 of SEQ ID NO: 5.
  • the region within amino acid 26 to 760 of SEQ ID NO: 5 specifically bound or targeted by the antibody fragment, or the VHH or fragment thereof, may be a linear region (i.e. linear epitope or sequential epitope) within said primary amino acid sequence. Alternatively said region may not be linear and may correspond to a conformational epitope.
  • a linear epitope comprises a linear sequence of amino acids that has a length of 5 to 30 amino acids, that is to say that it may have a length of 5, 6, 7, 8, 9 ,10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • a conformational epitope is characterized by a number of non-consecutive amino acids within amino acid 26 to 760 of SEQ ID NO: 5 that come together in the three-dimensional tertiary structure of the protein and that are contacted by the antibody fragment.
  • Second structural feature of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: based on the epitope of the antibody fragment.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human and/or murine FAP may alternatively or in combination with the first structural feature defined above also be further defined by a second structural feature defined below.
  • a second structural feature is that an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to a number of amino acids within amino acid 26 to 760 of SEQ ID NO: 5. These specific amino acids within amino acid 26 to 760 of SEQ ID NO: 5 are further defined below.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to at least one of amino acid comprised within 65-90 and/or 101-140 of SEQ ID NO: 5.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to at least one of amino acid I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91 , L105, S106, P107, D108, R109, Q110, F111 , D134, L135, S136, N137, V158, G159, R175, D457 and Y458 of SEQ ID NO: 5.
  • the stretch of amino acids 65-90 of SEQ ID NO: 1 defines a first region of hFAP, which is contacted, bound or specifically bound by the antibody fragment. Not each amino acid within this stretch or region may be contacted, bound or specifically bound by the antibody fragment. In an embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of this stretch or region is contacted, bound or specifically bound by the antibody fragment. In an embodiment, this first stretch or region is an epitope of the antibody fragment. In an embodiment, an epitope of the antibody fragment is comprised within this first stretch or region.
  • At least one of Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83, G84, Q85, S86, Y87, T88, I89, L90 is contacted, bound or specifically bound by the antibody fragment.
  • the stretch of amino acids 101-140 of SEQ ID NO: 5 defines a second region of hFAP, which is contacted, bound or specifically bound by the antibody fragment. Not each amino acid within this stretch or region needs to be contacted, bound or specifically bound by the antibody fragment. In an embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids of this stretch or region is contacted, bound or specifically bound by the antibody fragment. In an embodiment, this second stretch or region is an epitope of the antibody fragment. In an embodiment, an epitope of the antibody fragment is comprised within this second stretch or region.
  • At least one of L105, S106, P107, D108, R109, Q110, F111 , D134, L135, S136, N137 is contacted, bound or specifically bound by the antibody fragment.
  • the antibody fragment further contacts additional amino acids as I62, S63, G64, S91 , V158, G159, R175, D457 and/or Y458 of SEQ ID NO: 5.
  • each of the first and second stretches or regions defined above is contacted, bound or specifically bound by the antibody fragment.
  • the combination of these two stretches defines the conformational epitope of the antibody fragment.
  • a conformational epitope is comprised within the combination ofthese two stretches. Not each amino acid within each of these stretches or regions may be contacted, bound or specifically bound by the antibody fragment.
  • 1 , 2, 3, 4, 5 or 6 amino acids (or more depending on the length of each stretch) of each of the stretches or regions is contacted, bound or specifically bound by the antibody fragment.
  • the antibody fragment further contacts additional amino acids as I62, S63, G64, S91 , V158, G159, R175, D457 and/or Y458 of SEQ ID NO: 5.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human FAP wherein the epitope is comprised within the amino acid stretch or region 65-90 and/or 101-140 of SEQ ID NO: 5.
  • the antibody fragment, or the VHH or fragment thereof further contacts additional amino acids as I62, S63, G64, S91 , V158, G159, R175, D457 and/or Y458 of SEQ ID NO: 5.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human FAP wherein the epitope is comprised within the combination of amino acid stretches or regions 65-90 and 101-140 of SEQ ID NO: 5.
  • the antibody fragment, or the VHH or fragment thereof further contacts additional amino acids as I62, S63, G64, S91 , V158, G159, R175, D457 and/or Y458 of SEQ ID NO: 5.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 5:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 5:
  • the following amino acids of SEQ ID NO: 5 are bound or contacted or interacted with an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91 , L105, S106, P107, D108, R109, Q110, F111 , D134, L135, S136, N137, V158, G159, R175, D457 and Y458.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83, G84, Q85, S86, Y87, T88, I89, L
  • the following amino acids of SEQ ID NO: 5 are bound or contacted or interacted with an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: I62, S63, G64, Q65, E66, I76, V77, L78, N80, 181 , E82, T83, Q85, S86, Y87, T88, I89, L90, S91 , L105, S106, P107, D108, R109, Q110, F111 , D134, L135, S136, N137, V158, G159, R175, D457 and Y458.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: I62, S63, G64, Q65, E66, I76, V77, L78, N80, 181 , E82, T83, Q85, S86, Y87, T88, I89, L90, S91 , L105, S106, P107
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: based on the full length sequence
  • a third structural feature is that an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof relates to the full length amino acid sequence.
  • the present application discloses a family of structurally closely related antibody fragments represented by an amino acid sequence comprising, consisting of or essentially consisting of SEQ ID NO: 4 or a fragment thereof.
  • VHH VHH1 is represented by SEQ ID NO: 4 (see table below).
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be defined by its first structural feature as defined above and its third structural feature further defined below.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be defined by its second structural feature as defined above and its third structural feature further defined below.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4 or a portion thereof.
  • sequence identity with this sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 81 % sequence similarity with SEQ ID NO: 4 or a portion thereof.
  • the sequence similarity with this sequence is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 80% sequence identity with SEQ ID NO:4 or a portion thereof and has a length which is ranged from the exact length of SEQ ID NO: 4 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO:4.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 81% sequence similarity with SEQ ID NO:4 or a portion thereof and has a length which is ranged from the exact length of SEQ ID NO: 4 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO:4.
  • a tag such as a His-tag may be added to an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof.
  • His-tag comprises 4, 5, 6, 7, 8, 9, 10 Histidines.
  • Alternative tag may be a Hemagglutinin tag (HA-tag): YPYDVPDYA (SEQ ID NO: 11 ); YPYDVPDYGS (SEQ ID NO: 12) or a cysteine tag (Cys tag).
  • a cysteine tag is a tag that comprises one or several cysteines.
  • Non-limiting examples of cysteine tags are C; GGC; SPSTPPTPSPSTPPC (SEQ ID NO: 13)
  • identity and similarity are assessed is explained in detail in the part dedicated to definition at the end of the description. Usually when identity is defined by reference to a SEQ ID NO, said identity is assessed over the whole SEQ ID NO. However, it is also encompassed by the application that identity (or similarity) is assessed over a portion (or a fragment) of said sequence. Within this context, a portion may mean at least 50%, 60%, 70%, 80%, 90%, 95% of the length of the SEQ ID NO. The length of the sequence encompassed may still be longer than the length of the SEQ ID NO used to assess the identity (or similarity) (i.e.
  • length being at least 50% of the length of the SEQ ID NO, 60%, 70%, 80%, 90%, the same as the one of the SEQ ID NO even though the identity (or similarity) is assessed over a portion of this SEQ ID NO, or the length being 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO.
  • the length of the antibody fragment, or the VHH or fragment thereof is from 110 to 130 amino acids or 110, 111 , 112, 1 13, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.
  • This length does not include the length of a tag, such as a His tag that may be added to the sequence of the antibody fragment.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has a length which is ranged from the exact length of SEQ ID NO: 4 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO:4.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has a length which is ranged from 110 to 130 amino acids or 110, 111 , 112, 113, 114, 115, 116,
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 80% sequence identity with at least one of SEQ ID NO: 4 or a portion thereof and the length of the antibody fragment, or the VHH or fragment thereof, is from 80 to 150 amino acids or 90 to 140 or 100 to 130 or 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117,
  • sequence identity with at least of one of these sequences is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 81 % sequence similarity with at least one of SEQ ID NO: 4 or a portion thereof and the length of the antibody fragment, or the VHH or fragment thereof, is from 80 to 150 amino acids or 90 to 140 or 100 to 130 or 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.
  • sequence similarity with at least of one of these sequences is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts, binds or specifically binds at least one of (preferably both) the stretches or regions of amino acids of SEQ ID NO: 5 as defined earlier herein (i.e. amino acid stretch or region 65-90 and/or 101-140 of SEQ ID NO: 5).
  • an epitope of said antibody fragment is comprised within these stretches or regions of amino acids of SEQ ID NO: 5.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has for conformational epitope the combination of stretches or regions of amino acids of SEQ ID NO: 5 as defined earlier herein (i.e. amino acid stretches or regions 65-90 and/or 101-140 of SEQ ID NO: 5).
  • a conformational epitope of said antibody fragment is comprised within these stretches or regions of amino acids of SEQ ID NO: 5.
  • These epitopes define a family of antibody fragments. This family of antibody fragments shares at least one of these epitopes, linear epitopes and/or this conformational epitope.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof:
  • - is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 80% sequence identity (or similarity) with SEQ ID NO: 4 or a portion thereof (third structural feature) and
  • sequence identity (or similarity) with this sequence is at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has a length which is ranged from the exact length of SEQ ID NO: 4 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 4.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof:
  • - is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 80% sequence identity (or similarity) with SEQ ID NO: 4 or a portion thereof (third structural feature) and
  • sequence identity (or similarity) with this sequence is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has a length which is ranged from the exact length of SEQ ID NO: 4 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 4.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by an amino acid sequence that comprises at least one combination of CDR sequences chosen from the group comprising: a CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 , a CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2, and a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3.
  • One or two amino acids of said CDR1 , CDR2 and/or CDR3 may have been substituted by another one amino acid without substantially altering the activity of the obtained antibody fragment. The same holds for any of the frame work regions of the antibody fragment.
  • An activity of said variant is a specific binding activity as earlier defined herein.
  • 'substantial' may mean at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of said binding activity is still detectable compared to the activity of the antibody fragment, or the VHH or fragment thereof, with the initial CDR and/or FR regions.
  • the present application provides an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprising the heavy chain antibodies with the (general) structure or which is derived therefrom:
  • FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and are as further defined herein (see table 2 listing the amino acid sequences of heavy chain variable domains that have been raised against human and/or murine FAP).
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof (or of the nucleotide sequences to express these), nor as to the way that the antibody fragment, or the VHH or fragment thereof, or nucleotide sequences disclosed herein are (or have been) generated or obtained.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences.
  • the amino acid sequence of the antibody fragment, or the VHH or fragment thereof is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to “humanized” immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), “camelized” immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.
  • “humanized” immunoglobulin sequences such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized amino acid sequences of the invention.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is derived from the antibody fragment, or the VHH or fragment thereof, described above using CDR grafting.
  • a preferred antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof, comprises a:
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2
  • CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3.
  • FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 and a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3, FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment. CDR2 region is from another antibody fragment.
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2 and a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3, FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment. CDR1 region is from another antibody fragment.
  • FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment. CDR3 region is from another antibody fragment.
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 and it comprises a CDR2 and CDR3 regions from another antibody fragment, and may have the FR of as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2 and it comprises a CDR1 and CDR3 regions from another antibody fragment and may have the FR as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3 and it comprises a CDR1 and CDR2 regions from another antibody fragment and may have the FR as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • an amino acid sequence comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence)
  • said sequence may optionally be further suitably humanized, again as described herein, so as to provide one or more further (partially or fully) humanized amino acid sequences as disclosed herein.
  • humanized antibody fragment, or VHH or fragment thereof may be represented by amino acid sequences in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution.
  • other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring antibody fragment sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said antibody fragment sequence (in any manner known per se, as further described herein) and the resulting humanized antibody fragment sequences or functional fragments thereof can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be defined by its first structural feature as defined above and its fourth structural feature further defined herein.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be defined by its second structural feature as defined above and its fourth structural feature further defined herein.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be defined by its second structural feature as defined above, its third structural feature and its fourth structural feature further defined herein.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts, binds or specifically binds at least one of (preferably both) the stretches or regions of amino acids of SEQ ID NO: 5 as defined earlier herein (i.e. amino acid stretch or region 65-90 and/or 101-140 of SEQ ID NO: 5).
  • an epitope of said antibody fragment is comprised within these stretches or regions of amino acids of SEQ ID NO: 5.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may have for conformational epitope the combination of stretches or regions of amino acids of SEQ ID NO: 5 as defined earlier herein (i.e. amino acid stretches or regions 65-90 and/or 101-140 of SEQ ID NO: 5).
  • a conformational epitope of said antibody fragment is comprised within the combination of these stretches or regions of amino acids of SEQ ID NO: 5.
  • These epitopes define a family of antibody fragments. This family shares at least one of these epitopes, linear epitopes and/or this conformational epitope.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprises a:
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1
  • a CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2
  • a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3.
  • FR regions may be as identified in table 2.
  • FR regions may be from another antibody fragment.
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 and a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3
  • FR regions may be as identified in table 2.
  • FR regions may be from another antibody fragment.
  • CDR2 region is from another antibody fragment.
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2 and a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3, FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment. CDR1 region is from another antibody fragment.
  • FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment. CDR3 region is from another antibody fragment.
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 and it comprises a CDR2 and CDR3 regions from another antibody fragment and may have the FR of as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2 and it comprises a CDR1 and CDR3 regions from another antibody fragment and may have the FR as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3 and it comprises a CDR1 and CDR2 regions from another antibody fragment and may have the FR as identified in table 2.
  • FR regions may be from another antibody fragment (fourth structural feature) and
  • - has an epitope comprised within the amino acid stretch or region comprised within 65-90 and/or 101-140 of SEQ ID NO: 5 (second structural feature).
  • a preferred antibody fragment such as a single-domain antibody fragment, preferably VHH, or fragment thereof, comprises a:
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2
  • CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3.
  • FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 and a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3, FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment. CDR2 region is from another antibody fragment.
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2 and a CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3, FR regions may be as identified in table 2. Alternatively, FR regions may be from another antibody fragment. CDR1 region is from another antibody fragment.
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 and a CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2
  • FR regions may be as identified in table 2.
  • FR regions may be from another antibody fragment.
  • CDR3 region is from another antibody fragment.
  • CDR1 region comprising or consisting of or essentially consisting of SEQ ID NO: 1 and it comprises a CDR2 and CDR3 regions from another antibody fragment and may have the FR of as identified in table 2.
  • FR regions may be from another antibody fragment.
  • CDR2 region comprising or consisting of or essentially consisting of SEQ ID NO: 2 and it comprises a CDR1 and CDR3 regions from another antibody fragment, and may have the FR as identified in table 2. Alternatively, FR regions may be from another antibody fragment.
  • CDR3 region comprising or consisting of or essentially consisting of SEQ ID NO: 3 and it comprises a CDR1 and CDR2 regions from another antibody fragment and may have the FR as identified in table 2.
  • FR regions may be from another antibody fragment (fourth structural feature) and
  • Each of the other embodiments of the second structural feature may be combined with each embodiment of the fourth structural feature.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by a first and/or second and/or third and/or fourth structural feature as identified herein.
  • said antibody fragment is characterized by at least one of the following functional features:
  • an antibody fragment such as a singledomain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to human and/or murine FAP with a KD ranged from 10' 9 to 10' 12 moles/liter and/or a k O ff ranging from 10' 2 to 10' 5 s -1 preferably assessed using bio-layer interferometry, more preferably with a KD ranged from 10' 9 to 10' 12 moles/liter and a k O ff ranging from 10' 2 to 10' 5 s' 1
  • the second functional feature relating to the fact the antibody fragment, or the VHH or fragment thereof, may not be a modulator (i.e. is not an inhibitor, is not an activator of human and/or murine FAP) has also been described in detail herein.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof should therefore fulfil at least one of the structural features and/or at least one of the functional features.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof, wherein said antibody fragments are able to specifically bind human fibroblast activation protein (FAP), wherein said antibody fragments may be represented by a sequence comprising SEQ ID NO: 5, 6 and 7, preferably wherein said sequence has at least 80% sequence identity with SEQ ID NO: 8 or at least 80% sequence identity over 50% of the length of SEQ ID NO: 4; preferably wherein said labels comprise a chelator, more preferably wherein rLAR (i.e. rCAR) is from 0.50 to 0.75, even more preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • FAP human fibroblast activation protein
  • the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof which specifically bind human and/or murine FAP, wherein the epitope is comprised within the amino acid stretch or region 65-90 and/or 101-140 of SEQ ID NO: 5; and wherein said labels comprise a chelator, wherein rLAR (i.e. rCAR) from 0.50 to 0.75, preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof which specifically bind human and/or murine FAP, wherein the conformational epitope is comprised within the combination of amino acid stretch or region 65-90 and 101-140 of SEQ ID NO: 5; wherein said labels comprise a chelator, wherein rLAR (i.e. rCAR) is from 0.50 to 0.75, preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof which specifically bind human and/or murine FAP, wherein at least one of amino acids I62, S63, G64, Q65, E66, I76, V77, L78, Y79, N80, 181 , E82, T83, G84, Q85, S86, Y87, T88, I89, L90, S91 , L105, S106, P107, D108, R109, Q110, F111 , D134, L135, S136, N137, V158, G159, R175, D457 and/or Y458 of SEQ ID NO: 5 interacts with said VHH or fragment; wherein said labels comprise a chelator, wherein rLAR (i.e. rCAR) is from 0.50 to 0.75, preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • the chelator i.e.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof that specifically bind human and/or murine FAP, wherein said VHH or fragment is represented by an amino acid sequence having at least 80% sequence identity (or similarity) with at least one of SEQ ID NO: 1 , 2, 3, 4 or a portion thereof; wherein said labels comprise a chelator, wherein rLAR (i.e. rCAR) is from 0.50 to 0.75, preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof, wherein said antibody fragments are able to specifically bind human fibroblast activation protein (FAP), wherein said antibody fragments may be represented by a sequence comprising SEQ ID NO: 5, 6 and 7, preferably wherein said sequence has at least 80% sequence identity with SEQ ID NO: 8 or at least 80% sequence identity over 50% of the length of SEQ ID NO: 4; preferably wherein said labels comprise a chelator, more preferably wherein LAR (i.e. CAR) is from 2.00 to 3.00, even more preferably wherein the standard deviation of rLAR is equal to or lower than 1.00.
  • the chelator is DOTA.
  • populations according to the invention wherein the antibody fragments are able to specifically bind human fibroblast activation protein (FAP), and wherein rLAR (i.e. rCAR) is from 0.50 to 0.75, correspondingly wherein LAR (i.e. CAR) is from 2.00 to 3.00, have a higher melting point.
  • FAP human fibroblast activation protein
  • rLAR i.e. rCAR
  • LAR i.e. CAR
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is particularly suited for binding to human folate receptor alpha (FOLR1 ).
  • FOLR1 human folate receptor alpha
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds human folate receptor alpha (FOLR1 , which is represented by SEQ ID NO: 14), but does neither specifically bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) and is further characterized by an additional (structural) feature as defined herein (such as first, second, third and/or fourth structural features).
  • FOLR1 human folate receptor alpha
  • FOLR2 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds human folate receptor alpha (FOLR1 , which is represented by SEQ ID NO: 14), but does neither specifically bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) and is further characterized by an additional (structural) feature as defined herein (such as first, second, third and/or fourth structural features).
  • FOLR1 human folate receptor alpha
  • FOLR2 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds human folate receptor alpha (FOLR1 , which is represented by SEQ ID NO: 14), but does neither specifically bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) (or the antibody fragment specifically binds human FOLR1 but does neither specifically bind human FOLR2 nor human FOLR3) and fulfils at least one of the following: d. the epitope is comprised within amino acid 25 to 233 of SEQ ID NO: 14, e.
  • the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 60% sequence identity with at least one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22 or a portion thereof.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) (or the antibody fragment specifically binds human FOLR1 but does neither specifically bind human FOLR2 nor human FOLR3) and the antibody fragment thereof fulfils a) and/or b): a. the epitope is comprised within amino acid 25 to 233 of SEQ ID NO: 14 and the antibody fragment thereof specifically binds to the following amino acids of SEQ ID NO: 14:
  • the antibody fragment thereof is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), (or the antibody fragment specifically binds human FOLR1 but does neither specifically bind human FOLR2 nor human FOLR3), wherein the epitope is comprised within amino acid 25 to 233 of SEQ ID NO: 14 and the antibody fragment specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • Folate receptor alpha also known as folate receptor 1 or folate binding protein is denominated using the abbreviation FOLR1 or FR-alpha at the protein and at the gene levels.
  • FOLR1 Folate receptor 1
  • FR-alpha is the recommended protein name from Uniprot and FOLR1 is used for the gene name.
  • the binding to human FOLR1 may be assessed in a homogeneous mixture of different antigens.
  • a specific binding interaction will discriminate between desirable and undesirable antigens in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000- fold).
  • the binding may be assessed in vitro using cells expressing human FOLR1 , and optionally in vivo or ex vivo as earlier defined herein. These cells may be human cells and expressing endogenous human FOLR1. Alternatively, these cells may overexpress human FOLR1. Cells overexpressing human FOLR1 may be human or non-human cells. Preferred cells are SKOV3 and OVCAR3.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to human FOLR1 and does not specifically bind to murine FOLR1 or to human FOLR2 or to human FOLR3.
  • This assessment is preferably carried out using ELISA or SPR.
  • the antibody fragment will bind to a number of naturally occurring or synthetic analogues, variants, mutants, alleles, parts and fragments of human FOLR1.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof will specifically bind to at least those analogues, variants, mutants, alleles, naturally occurring, synthetic analogues, parts and fragments of human FOLR1 that (still) contain the epitope of the (natural/wild-type) antigen to which the antibody fragment binds.
  • the epitope of human FOLR1 for the antibody fragment is comprised within amino acid 25 to 233 of SEQ ID NO: 14.
  • At least one of the following amino acids of this part of SEQ ID NO: 14 is bound by the antibody fragment: C89, G90, E91 , M92, A93, P94, E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181 , F182, P183 and/or T184 of SEQ ID NO: 14.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds to the following amino acids of SEQ ID NO: 14:
  • amino acids of amino acids 25 to 233 of SEQ ID NO: 14 are bound by an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: C89, G90, E91 , M92, A93, P94, E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181 , F182, P183 and T184.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: C89, G90, E91 , M92, A93, P94, E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181 , F182, P183 and T184.
  • an amino acid of human FOLR1 may be bound by the antibody fragment when said amino acid belongs to the epitope of the antibody fragment.
  • a first structural feature is that an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to a region of human FOLR1 comprised within amino acid 25 to 233 of SEQ ID NO: 14.
  • SEQ ID NO: 14 is the human amino acid sequence of human FOLR1.
  • Amino acid 25 to 233 (R25-M233) of SEQ ID NO: 14 is defined as the extracellular domain (without signal peptide M1-T24 and GPI anchor S234 and pro-peptide G235-S257) of human FOLR1 (UniProt Knowledgebase: entry P15328).
  • the region within amino acid 25 to 233 of SEQ ID NO: 14 specifically bound or targeted by the antibody fragment may be a linear region (i.e. linear epitope or sequential epitope) within said primary amino acid sequence.
  • said region may not be linear and may correspond to a conformational epitope.
  • a linear epitope comprises a linear sequence of amino acids that has a length of 5 to 30 amino acids, that is to say that it may have a length of 5, 6, 7, 8, 9 ,10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • a conformational epitope is characterized by a number of non-consecutive amino acids within amino acid 25 to 233 of SEQ ID NO: 14 that come together in the three-dimensional tertiary structure of the protein and that are contacted by the antibody fragment.
  • linear epitopes and conformational epitope of the antibody fragment are defined.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) may alternatively or in combination also be further defined by a second structural feature.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) may alternatively or in combination also be further defined by a second structural feature.
  • a single-domain antibody fragment preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1)
  • FOLR2 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • a second structural feature is that an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to a number of amino acids within amino acid 25 to 233 of SEQ ID NO: 14. These specific amino acids within amino acid 25 to 233 of SEQ ID NO: 14 are further defined below.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to at least one of amino acid C89, G90, E91 , M92, A93, P94, E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181 , F182, P183 and/or T184 of SEQ ID NO: 14.
  • Each combination of 2, 3, 4, 5, 6,7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or 26 amino acids from the 26 amino acids identified is therefore encompassed to be contacted by the antibody fragment.
  • the linear stretch of amino acids 89-94 of SEQ ID NO: 14 defines a first linear region of hFOLRI , which is contacted, bound or specifically bound by the antibody fragment. Not each amino acid within this stretch or region may be contacted, bound or specifically bound by the antibody fragment. In an embodiment, 1 , 2, 3, 4, 5 or 6 amino acids of this linear stretch or region is contacted, bound or specifically bound by the antibody fragment. In an embodiment, this first linear stretch or region is an epitope of the antibody fragment.
  • the linear stretch of amino acids 140-150 of SEQ ID NO: 14 defines a second linear region of hFOLRI , which is contacted, bound or specifically bound by the antibody fragment. Not each amino acid within this stretch or region needs to be contacted, bound or specifically bound by the antibody fragment. In an embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids of this linear stretch or region is contacted, bound or specifically bound by the antibody fragment. In an embodiment, this second linear stretch or region is a second epitope of the antibody fragment.
  • the linear stretch of amino acids 176-184 of SEQ ID NO: 14 defines a third linear region of hFOLRI , which is contacted, bound or specifically bound by the antibody fragment. Not each amino acid within this stretch or region needs to be contacted, bound or specifically bound by the antibody fragment. In an embodiment, 1 , 2, 3, 4, 5, 6, 7, 8 or 9 amino acids of this linear stretch or region is contacted, bound or specifically bound by the antibody fragment. In an embodiment, this third linear stretch or region is a third epitope of the antibody fragment. In an embodiment, each of the first, second and third linear stretches or regions defined above is contacted, bound or specifically bound by the antibody fragment. The combination of these three stretches defines the conformational epitope of the antibody fragment.
  • each amino acid within each of these stretches or regions may be contacted, bound or specifically bound by the antibody fragment.
  • 1 , 2, 3, 4, 5 or 6 amino acids (or more depending on the length of each stretch) of each of the linear stretches or regions is contacted, bound or specifically bound by the antibody fragment.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the linear amino acid stretch or region 89-94, 140-150 and/or 176-184 of SEQ ID NO: 14.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the linear amino acid stretch or region 89-94, 140-150 and/or 176-184 of SEQ ID NO: 14.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the combination of linear amino acid stretches of regions 89-94, 140-150 and 176- 184 of SEQ ID NO: 14.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the combination of linear amino acid stretches of regions 89-94, 140-150 and 176-184 of SEQ ID NO: 14.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14: at least one, or at least two, or at least three amino acids selected from C89, G90, E91 , M92, A93, or P94, and/or at least one, or at least two, or at least three amino acids selected from E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, or Y150, and/or at least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181 , F182, P183, or T184.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14: at least one, or at least two, or at least three, or at least four, or at least five or all six amino acids selected from C89, G90, E91 , M92, A93, or P94, and at least one, or at least two, or at least three, or at least four, or at least five, or at least six or at least seven, or at least eight or at least nine or at least ten or all eleven amino acids selected from E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, or Y150, and at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or all nine amino acids selected from Q176, P177, F178, H179, F180,
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14:
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14: C89, G90, E91 , M92, A93 and/or P94.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14: E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149 and/or Y150.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14: Q176, P177, F178, H179, F180, Y181 , F182, P183 and/or T184.
  • amino acids of amino acids 25 to 233 of SEQ ID NO: 14 are contacted, bound or specifically bound by an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: C89, G90, E91 , M92, A93, P94, E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181 , F182, P183 and T184.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof: C89, G90, E91 , M92, A93, P94, E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, Y150, Q176, P177, F178, H179, F180, Y181 , F182, P183 and T184.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) may alternatively or in combination also be further defined by a third structural feature.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) may alternatively or in combination also be further defined by a third structural feature.
  • a third structural feature relates to the (full length) amino acid sequence representing a way of defining the family of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof.
  • the present application discloses a family of structurally closely related antibody fragments represented by an amino acid sequence comprising, consisting of or essentially consisting of SEQ ID NO: 21 or 22.
  • VHH VHH2 is represented by SEQ ID NO: 21 and VHH3 by SEQ ID NO: 22 (see table 4 below).
  • SEQ ID NO: 15, 17 and 18 is a part of VHH2 which is conserved amongst the family of the antibody fragment (see table 4 below).
  • SEQ ID NO: 16, 19 and 20 is a part of VHH3 which is conserved amongst the family of the antibody fragment (see table 4 below).
  • VHHs VHH2 and VHH3 both contact, bind or specifically bind each of the linear stretches or regions of amino acids of SEQ ID NO: 14 as defined earlier herein (i.e. linear amino acid stretch or region 89-94, 140-150 and/or 176-184 of SEQ ID NO: 14). Moreover, both VHH2 and VHH3 have for conformational epitope the combination of stretches or regions of amino acids of SEQ ID NO: 14 as defined earlier herein (i.e. linear amino acid stretches or regions 89- 94, 140-150 and 176-184 of SEQ ID NO: 14). VHH2 and VHH3 are two members of a family of antibody fragments. This family of antibody fragments shares at least one of these linear epitopes and/or this conformational epitope.
  • This third structural feature of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be defined in several ways:
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with at least one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22 or a portion thereof.
  • sequence identity with at least of one of these sequences is at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with at least one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22 or a portion thereof and has a length which is ranged from the exact length of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22 or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22.
  • a tag such as a His tag may be added to the antibody fragment. Usually His tag comprises 4, 5, 6, 7, 8, 9, 10 histidines.
  • Alternative tag may be a Hemaglutinin tag (HA-tag): YPYDVPDYA (SEQ ID NO: 37); YPYDVPDYGS (SEQ ID NO: 38) or a cysteine tag (Cys tag).
  • a cysteine tag is a tag that comprises one or several cysteine. Non-limiting examples of cysteine tags are C; GGC; SPSTPPTPSPSTPPC (SEQ ID NO: 39)
  • identity and similarity are assessed is explained in detail in the part dedicated to definition as the end of the description. Usually when identity is defined by reference to a SEQ ID NO, said identity is assessed over the whole SEQ ID NO. However, it is also encompassed by the application that identity (or similarity) is assessed over a portion (or a fragment) of said sequence. Within this context, a portion may mean at least 50%, 60%, 70%, 80%, 90%, 95% of the length of the SEQ ID NO. The length of the sequence encompassed may still be longer than the length of the SEQ ID NO used to assess the identity (or similarity) (i.e.
  • length being at least 50% of the length of the SEQ ID NO, 60%, 70%, 80%, 90%, the same as the one of the SEQ ID NO even though the identity(or similarity) is assessed over a portion of this SEQ ID NO, or the length being 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 amino acids longer than the exact length of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22).
  • the length of the antibody fragment is from 110 to 130 amino acids or 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.
  • This length does not include the length of a tag, such as a His tag that may be added to the sequence of the antibody fragment.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with at least one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 or 22 or a portion thereof and the length of the antibody fragment is from 80 to 150 amino acids or 90 to 140 or 100 to 130 or 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.
  • sequence identity (or similarity) with at least of one of these sequences is at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 15 or a portion thereof.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 15 or a portion thereof and has a length which is ranged from 78 to 130 amino acids or 78 to 130 or 90 to 120 or 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120,
  • sequence identity is at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Portion thereof has been already defined herein.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 17 and/or 18 or a portion thereof.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with any of SEQ ID NO: 17 and/or 18 or a portion thereof and has a length which is ranged from 14 to 130 amino acids or 20 to 120 or 30 to 110 or 14, 15, 16, 17, 18, 19, 20, 21 , 22,
  • sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Portion thereof has been already defined herein.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 16 or a portion thereof.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with any of SEQ ID NO: 16 or a portion thereof and has a length which is ranged from 76 to 130 amino acids or 76 to 120 or 90 to 110 or 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117,
  • sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Portion thereof has been already defined herein.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 19 and/or 20 or a portion thereof.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 19 and/or 20 or a portion thereof and has a length which is ranged from 13 to 150 amino acids or 13 to 140 or 30 to 130 or 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 ,
  • sequence identity is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Portion thereof has been already defined herein.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 21 and/or 22 or a portion thereof.
  • the antibody fragment is represented by an amino acid sequence that comprises or essentially consists of an amino acid sequence having at least 60% sequence identity (or similarity) with SEQ ID NO: 21 and/or 22 or a portion thereof and has a length which is ranged from 110 to 130 amino acids or 100 to 130 or 110 to 120 or 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.
  • sequence identity is at least 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Portion thereof has been already defined herein.
  • the antibody fragment is represented by an amino acid sequence that comprises any of SEQ ID NO: 15, 16, 17, 18, 19 and/or 20. In an embodiment, it has a length which is ranged from 13 to 130 amino acids or 20 to 120 or 30 to 121 amino acids. More preferably it is represented by an amino acid sequence that comprises SEQ ID NO: 21 or 22. Even more preferably, it has a length of 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129 or 130 amino acids.
  • the antibody fragment is according to the ninth embodiment of this third structural feature and contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14: at least one, or at least two, or at least three amino acids selected from C89, G90, E91 , M92, A93, or P94, and/or at least one, or at least two, or at least three amino acids selected from E140, Q141 , W142, W143, E144, D145, C146, R147, T148, S149, or Y150, and/or at least one, or at least two, or at least three amino acids selected from Q176, P177, F178, H179, F180, Y181 , F182, P183, or T184.
  • the antibody fragment is according to the tenth embodiment of this third structural feature and contacts or binds or specifically binds to the following amino acids of SEQ ID NO: 14: at least one, or at least two, or at least three amino acids selected from C89, G90, E91 , M92, A93, or P94, and/or at least one, or at least two, or at least three amino acids selected from E140, Q141 , W142, W143,
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • sequence identity is at least 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the linear amino acid stretch or region 89-94, 140-150 and/or 176-184 of SEQ ID NO: 14 and wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • FOLR1 human folate receptor alpha
  • FOLR2 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the linear amino acid stretch or region 89-94, 140-150 and/or 176-184 of SEQ ID NO: 14 and wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • FOLR1 human folate receptor alpha
  • FOLR2 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • sequence identity is at least 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the combination of linear amino acid stretches or regions 89-94, 140-150 and 176-184 of SEQ ID NO: 14, and wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • FOLR1 human folate receptor alpha
  • FOLR2 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3), wherein the epitope comprises the combination of linear amino acid stretches or regions 89-94, 140-150 and 176-184 of SEQ ID NO: 14, and wherein the antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence.
  • FOLR1 human folate receptor alpha
  • FOLR2 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • sequence identity is at least 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) may also be alternatively or in combination further defined by a fourth structural feature.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof that specifically binds an epitope of human folate receptor alpha (FOLR1 ), but does neither bind human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) may also be alternatively or in combination further defined by a fourth structural feature.
  • the antibody fragment is represented by an amino acid sequence that comprises at least one combination of CDR sequences chosen from the group comprising: a CDR1 region having SEQ ID NO: 23, a CDR2 region having has SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 25, and/or a CDR1 region having SEQ ID NO: 30 a CDR2 region having has SEQ ID NO: 31 , and a CDR3 region having SEQ ID NO: 32 (see table 5).
  • the present application provides heavy chain variable domains comprising the heavy chain antibodies with the (general) structure or which is derived therefrom:
  • FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and are as further defined herein.
  • SEQ ID NO: 21 and 22 (see table 5) give the amino acid sequences of heavy chain variable domains that have been raised against human FOLR1.
  • the application is not limited as to the origin of the antibody fragment (or of the nucleotide sequences to express these), nor as to the way that the antibody fragments, preferably VHH or fragments thereof or nucleotide sequences disclosed herein are (or have been) generated or obtained.
  • the antibody fragments may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences.
  • the amino acid sequence of the antibody fragment is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to “humanized” immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), “camelized” immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be suitably humanized, as further described herein, so
  • the antibody fragment is derived from the antibody fragment described above using CDR grafting.
  • Preferred antibody fragments comprise a:
  • CDR1 region having SEQ ID NO: 23 a CDR2 region having has SEQ ID NO: 31 , and a CDR3 region having SEQ ID NO: 25,
  • CDR1 region having SEQ ID NO: 23 a CDR2 region having has SEQ ID NO: 31 , and a CDR3 region having SEQ ID NO: 32,
  • CDR1 region having SEQ ID NO: 23 a CDR2 region having has SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 32
  • CDR1 region having SEQ ID NO: 30 a CDR2 region having has SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 32
  • CDR1 region having SEQ ID NO: 30 a CDR2 region having has SEQ ID NO: 24, and a CDR3 region having SEQ ID NO: 25, or
  • CDR1 region having SEQ ID NO: 30 a CDR2 region having has SEQ ID NO: 31 , and a CDR3 region having SEQ ID NO: 25.
  • Each of these antibody fragments may have the FR of VHH2 or of VHH3 as identified in table 5. However, distinct FR may be present.
  • the antibody fragment is derived from the antibody fragment described above using CDR grafting.
  • Preferred antibody fragments thereof comprise a:
  • Each of these antibody fragments may comprise a CDR2 region from another antibody fragment and may have the FR of VHH2 or of VHH3 as identified in table 5. However, distinct FR may be present.
  • the antibody fragment is derived from the antibody fragment described above using CDR grafting.
  • Preferred antibody fragments comprise a:
  • Each of these antibody fragments may comprise a CDR3 region from another antibody fragment and may have the FR of VHH2 or of VHH3 as identified in table 5. However, distinct FR may be present.
  • the antibody fragment is derived from the antibody fragment described above using CDR grafting.
  • Preferred antibody fragments comprise a:
  • Each of these antibody fragments may comprise a CDR1 region from another antibody fragment and may have the FR of VHH2 or of VHH3 as identified in table 5. However, distinct FR may be present.
  • the antibody fragment is derived from the antibody fragment described above using CDR grafting.
  • Preferred antibody fragments comprise a:
  • CDR1 region having SEQ ID NO: 23 or 30 may comprise a CDR2 and CDR3 region from another antibody fragment and may have the FR of VHH2 or of VHH3 as identified in table 5. However, distinct FR may be present.
  • the antibody fragment is derived from the antibody fragment described above using CDR grafting.
  • Preferred antibody fragments comprise a:
  • CDR2 region having SEQ ID NO: 24 or 31 may comprise a CDR1 and CDR3 region from another antibody fragment and may have the FR of VHH2 or of VHH3 as identified in table 5. However, distinct FR may be present.
  • the antibody fragment is derived from the antibody fragment described above using CDR grafting. Preferred antibody fragments comprise a:
  • CDR3 region having SEQ ID NO: 25 or 19 may comprise a CDR1 and CDR2 region from another antibody fragment and may have the FR of VHH2 or of VHH3 as identified in table 5. However, distinct FR may be present.
  • an amino acid sequence comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence)
  • said sequence may optionally be further suitably humanized, again as described herein, so as to provide one or more further (partially or fully) humanized amino acid sequences as disclosed herein.
  • humanized antibody fragments may be represented by amino acid sequences in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution.
  • other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences or functional fragments thereof can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by a first, second, third and/or fourth structural feature as identified herein.
  • the antibody fragment is characterized by a functional feature which is to specifically bind human folate receptor alpha (FOLR1 ), but (preferably not murine FOLR1 ), not human folate receptor beta (FOLR2) and not human folate receptor gamma (FOLR3).
  • the antibody fragment is characterized by a functional feature which is to specifically bind human folate receptor alpha (FOLR1 ), but (preferably not murine FOLR1 ), not human folate receptor beta (FOLR2) and not human folate receptor gamma (FOLR3).
  • FOLR1 human folate receptor alpha
  • FOLR2 human folate receptor alpha
  • FOLR3 human folate receptor beta
  • FOLR3 human folate receptor gamma
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof that specifically bind an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) (or an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binding human FOLR1 , but neither binding human FOLR2 nor human FOLR3, wherein said labels comprise a chelator, preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof, wherein said antibody fragments are able to specifically bind human folate receptor alpha (FOLR1 ), wherein said antibody fragments may be represented by a sequence comprising SEQ ID NO: 22, 23 and 24, preferably wherein said sequence has at least 80% sequence identity with SEQ ID NO: 25 or at least 80% sequence identity over 50% of the length of SEQ ID NO: 17, preferably wherein said labels comprise a chelator, more preferably wherein the standard deviation of rLAR is equal to or lower than 0.25. Most preferably, the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof that specifically bind an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) (or an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binding human FOLR1 , but neither binding human FOLR2 nor human FOLR3), wherein the epitope comprises the combination of linear amino acid stretches or regions 89-94, 140-150 and 176- 184 of SEQ ID NO: 14; wherein said labels comprise a chelator, preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof that specifically bind an epitope of human folate receptor alpha (FOLR1 ), but does neither bind murine FOLR1 nor human folate receptor beta (FOLR2) nor human folate receptor gamma (FOLR3) (or an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof specifically binding human FOLR1 , but neither binding human FOLR2 nor human FOLR3), wherein said antibody fragment is represented by an amino acid sequence that comprises an amino acid sequence having at least 63% sequence identity with at least one of SEQ ID NO: 21 , 22, 15, 16, 17, 18, 19, or 20 over the full length of said sequence or over at least 50% of the length of said sequence, wherein said labels comprise a chelator, preferably wherein the standard deviation of rLAR is equal to or lower than 0.25.
  • the chelator is DOTA.
  • HER2 Human epidermal growth factor receptor 2
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is particularly suited for binding to human epidermal growth factor receptor 2.
  • the antibody fragment does not compete with the monoclonal antibody T rastuzumab (Herceptin®) or the monoclonal antibody Pertuzumab (Perjeta®) for binding to HER2, as determined using a suitable competition assay.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprises one of the CDR combinations chosen from the group comprising: a CDR1 region having SEQ ID NO: 40, a CDR2 region having SEQ ID NO: 41 , and a CDR3 region having SEQ ID NO: 42, and/or a CDR1 region having SEQ ID NO: 43, a CDR2 region having SEQ ID NO: 44, and a CDR3 region having SEQ ID NO: 45.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% amino acid identity with at least one of the amino acid sequences of SEQ ID NO: 46 or 47.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprises amino acid sequences SEQ ID NO: 46 or 47.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprises or consists of any of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 60% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 70% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 80% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88. In embodiments, an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 90% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88. In embodiments, an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 95% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof comprises or consists of any of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 60% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 70% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 80% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88. In embodiments, an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 90% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88. In embodiments, an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof has at least 95% amino acid identity with at least one of amino acid sequences SEQ ID NO: 48-88.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is present in a monovalent format.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is devoid of a cysteine-containing tag, preferably a GGC-tag.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is non-lifetime extended.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is devoid of a carboxy-terminal polypeptide tag, preferably wherein said antibody fragment thereof is untagged.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof, wherein said antibody fragments are able to specifically bind human epidermal growth factor receptor 2 (HER2), wherein said antibody fragments may be represented by a sequence comprising SEQ ID NO: 40, 41 and 42, preferably wherein said sequence has at least 80% sequence identity with SEQ ID NO: 43 or at least 80% sequence identity over 50% of the length of SEQ ID NO: 43, wherein said labels comprise a chelator, preferably wherein the rLAR is from 0.43 to 0.57, more preferably wherein the standard deviation of rLAR is equal to or lower than 0.25. Most preferably, the chelator is DOTA.
  • the chelator is DOTA.
  • a population according to the invention comprises: antibody fragments such as a single-domain antibody fragments, preferably VHH, or fragments thereof, wherein said antibody fragments are able to specifically bind human epidermal growth factor receptor 2 (HER2), wherein said antibody fragments may be represented by a sequence comprising SEQ ID NO: 40, 41 and 42, preferably wherein said sequence has at least 80% sequence identity with SEQ ID NO: 43 or at least 80% sequence identity over 50% of the length of SEQ ID NO: 43, wherein said labels comprise a chelator, preferably wherein the rLAR is from 3.00 to 4.00, more preferably wherein the standard deviation of rLAR is equal to or lower than 4.00. Most preferably, the chelator is DOTA.
  • the chelator is DOTA.
  • populations according to the invention wherein the antibody fragments are able to specifically bind human epidermal growth factor receptor 2 (HER2), and wherein rLAR (i.e. rCAR) is from 0.43 to 0.57, correspondingly wherein LAR (i.e. CAR) is from 3.00 to 4.00, have a higher melting point.
  • rLAR i.e. rCAR
  • LAR i.e. CAR
  • composition comprising, consisting of, or consisting essentially of a population of antibody fragments according to the invention.
  • a composition comprising, consisting of, or consisting essentially of a population of antibody fragments according to the invention.
  • Such a composition is called a composition according to the invention in the context of this application. All preferred embodiments relating to a population, a label and an antibody fragment apply mutatis mutandis to such a composition.
  • compositions above may comprises an excipient.
  • the excipient should be acceptable for diagnostic and/or therapeutic purpose.
  • the composition is a pharmaceutical composition.
  • the composition is a diagnostic composition. It is also encompassed by the invention that the composition is a pharmaceutical and diagnostic composition. Suitable formulations of the invention are disclosed in the definition part at the end of the description.
  • the compositions may comprise one or more acceptable carrier.
  • the compositions may comprise at least one other compound.
  • the pharmaceutical compositions as envisaged herein can be used in the prevention and/or treatment of a cancer.
  • a method wherein a population according to the invention or a composition according to the invention is used to assess expression of an antigen, preferably human FAP or human FOLR1 , in a subject or in an isolated sample of said subject.
  • This method may comprise the following steps: a) providing a population according to the invention or a composition according to the invention, b) administering said population or composition to a subject in need thereof or to an isolated sample of a subject, c) assessing the expression of the antigen in said subject or in said isolated sample of said subject.
  • This method may be called a diagnostic method.
  • This method may be an in vitro or an in vivo method.
  • This method may allow the localization of the expression of the antigen, preferably human FAP or human FOLR1 or human HER2, in a subject or in an isolated sample of said subject and may allow the prediction and/or prognosis of a certain disease and/or disorder and/or condition in said subject.
  • this method may be a stratification method to identify patients that are likely to respond to a particular treatment such as cancer treatment or wound healing treatment or fibrosis treatment. Therefore, in a further aspect, there is provided a method wherein the population or composition according to the invention is used to stratify the subject and assess whether the subject will be likely to respond to a particular treatment such as cancer treatment or wound healing treatment or fibrosis treatment.
  • This method may comprise the following steps: a) providing a population according to the invention or a composition according to the invention, b) administrating said population or composition to a subject in need thereof or to an isolated sample of a subject, c) assessing the expression of the antigen in said subject or in said isolated sample of said subject and d) deciding whether the subject is likely to be responsive to a medical treatment.
  • the label is a radionuclide.
  • the subject is preferably a human being.
  • Detailed information is disclosed in the definition part at the end of the description in order to produce/provide and in order to administer a population according to the invention or a composition according to the invention.
  • a method according to this aspect may be an in vitro, ex vivo method.
  • a screening dose or a biomarker dose is administered to a subject or to an isolated sample of said subject. Detailed definitions are provided later on especially by comparison to the definition of a therapeutic dose.
  • an antigen preferably human FAP or human FOLR1 or human HER2
  • the assessment of the expression of an antigen, preferably human FAP or human FOLR1 or human HER2, in the subject is preferably carried out using an isolated sample of the subject.
  • an isolated sample of a subject may be a tissue or a liquid sample from said subject.
  • a liquid may be serum.
  • An isolated sample from a patient may be called a biopsy or a tumour biopsy.
  • a population according to the invention or a composition according to the invention is for medical use, preferably for use in the treatment and/or diagnosis of a cancer.
  • a population according to the invention or a composition according to the invention is for use in the treatment and/or diagnosis of a solid cancer, more preferably wherein said solid cancer is derived from an epithelial cell or tissue.
  • a population according to the invention or a composition according to the invention comprises a label which is a molecule to be delivered to a cell, a tissue, organ expressing or over-expressing the corresponding antigen, preferably human FAP or human FOLR1 or human HER2.
  • the molecule may be a peptide or a small molecule, a nucleic acid.
  • a peptide may be a cytokine.
  • a small molecule may be a chemotherapeutic.
  • a label may be a medicament for treating a disease or condition associated with the expression or the over-expression of the antigen, preferably FAP or FOLR1 or HER2.
  • a population according to the invention or a composition according to the invention is for use in the treatment of a cancer.
  • said cancer is associated with an expression of human FAP on a cancer or a tumour cell or a metastasized lesion.
  • the cancer treated may be metastatic, preferably wherein a metastatic cell is found in the brain, bones, liver, lung.
  • a cancer associated with expression of FAP may be any of a leukemia, bone, uterus, pancreas, GEP-NET (gastroenteropancreatic neuroendocrine tumour), skin, muscle, brain, breast, colorectal, esophageal, gastric, liver, lung, NSCLC (non small cell lung cancer) ovarian, parathyroid, renal cancer cells, CUP (cancer of unknown primary), prostate, small intestine, CCC (Cholangiocellular Carcinoma), sarcoma, (Pure et al 2018, Oncogene Aug;37(32):4343-4357 and Frederik Giesel et al J Nucl Med May 1 , 2019 vol. 60 no. supplement 1 Abstract 289).
  • the invention is not limited to these types of cancer.
  • the population or composition according to the invention may be used.
  • said cancer is associated with an expression of human FOLR1.
  • the label is a molecule to be delivered to the central nervous system (CNS).
  • the molecule preferably does not cross the brain blood barrier (BBB) on its own.
  • the molecule may be a peptide or a small molecule, a nucleic acid.
  • a peptide may be a cytokine.
  • a small molecule may be a chemotherapeutic.
  • a label may be a medicament for treating a disease or condition associated with the brain or wherein the medicament is for treating a disease or condition wherein an alteration of a brain activity will impact another organ of the subject.
  • the molecule is a medicament acting in the CNS, preferably acting in the brain. Even more preferably, the molecule crosses the brain blood barrier (BBB) and/or the blood-cerebrospinal fluid barrier (BCSFB) via transport via the human FOLR1 ; a mechanism called receptor mediated transcytosis (RMT). Any medicament known to act in the CNS or in the brain is potentially encompassed by the present invention as a label.
  • BBB brain blood barrier
  • BCSFB blood-cerebrospinal fluid barrier
  • RMT receptor mediated transcytosis
  • a disease or a condition or a disorder associated with the CNS or the brain may be any disease, condition or disorder known to the skilled person as being associated with the CNS or the brain. Such disease, condition or disorder may reflect an altered CNS activity or an altered brain activity. Examples of such disease, condition or disorder include: epilepsy, autism spectrum disorders, autism, altered food intake, altered heat regulation, altered pain sensation, chronic pain, depression, migraines, hearing loss, bipolar disorders, Alzheimer’s disease, schizophrenia, brain injury, blindness, stroke, Parkinson’s disease, multiple sclerosis, spinal cord injury, amyotrophic lateral sclerosis.
  • the cancer is associated with an expression of human FOLR1 on a cancer or a tumour cell or a metastasized lesion.
  • the cancer treated may be metastatic, preferably wherein a metastatic cell is found in the brain. This is an attractive embodiment as the labelled antibody fragments are able to cross the BBB.
  • a cancer associated with expression of FOLR1 may be any of an ovarian, endometrial, brain, lung, adrenal carcinoma, head and neck, breast, stomach, colon-rectum cancer. However, the invention is not limited to these types of cancer. As soon as a subject is suspected to have a cancer cell expressing or overexpressing human FOLR1 , a population according to the invention or a composition according to the invention may be used.
  • the subject has been first diagnosed using a population according to the invention or a composition according to the invention before being treated with a population or composition comprising the same or a different label, preferably a radionuclide.
  • a radionuclide may not be the same in diagnostic and therapy applications.
  • a disease or condition or disorder has been prevented or treated when the administration of a population according to the invention or a composition according to the invention has been carried out and has resulted: in the improvement of at least one symptom associated with said disease or condition or disorder and/or in the improvement of at least one parameter associated with said disease or condition or disorder.
  • the improvement may be observed at least one day, two days, three days, four days, five days, six days, one week after the population or composition has been administrated. Alternatively, the improvement may be observed at least one month, six months after the administration of the population or composition. Envisaged doses and administration modes are further disclosed in the definition part at the end of the description.
  • the (therapeutic) use of a population according to the invention or a composition according to the invention leads to or exhibits an anti-cancer activity when at least one of the following is fulfilled: it can kill a tumour cell, a cancer cell and/or a CAF that expresses the antigen, preferably human FAP or human FOLR1 or human HER2,
  • tumour cell or cancer cell It can reduce or slow the growth and/or proliferation of such a tumour cell or cancer cell.
  • An anti-cancer activity may have been identified or determined when the number of viable cancer cells, and/or viable tumour cells after the administration of the population or composition according to the invention is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% of the number of initial viable cancer cells and/or initial viable tumour cells.
  • An anti-cancer activity may have been identified or determined when the size of a primary tumour and/or the size of a metastatic lesion after the administration of the population or composition according to the invention is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% of the size of said primary tumour and/or of the size of said metastatic lesion.
  • Tumour cell death may be assessed by measurement of radiolabelled Annexin A5, a molecular imaging agent to measure cell death in vitro, and non-invasively in patients with cancer such as ICH (Schutters K. et al., Apoptosis 2010; de Saint-Hubert M. et al., Methods 48: 178, 2009).
  • ICH has been defined in the definition part at the end of the description.
  • Tumour growth may be inhibited at least 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more.
  • Tumour growth may be assessed using techniques known to the skilled person. Tumour growth may be assessed using MRI (Magnetic Resonance Imaging) or CT (Computer Tomography).
  • tumour weight increase or tumour growth may be inhibited at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70% or 75%, or more.
  • Tumour weight or tumour growth may be assessed using techniques known to the skilled person.
  • the detection of tumour growth or the detection of the proliferation of tumour cells may be assessed in vivo by measuring changes in glucose utilization by positron emission tomography with the glucose analogue 2-[18F]-fluor-2-deoxy-D-glucose (FDG-PET) or [18F]-'3-fluoro-'3-deoxy-L-thymidine (FLT-PET).
  • FDG-PET glucose analogue 2-[18F]-fluor-2-deoxy-D-glucose
  • FLT-PET [18F]-'3-fluoro-'3-deoxy-L-thymidine
  • An ex vivo alternative may be staining of a tumour biopsy with Ki67.
  • a delay in occurrence of metastases and/or of tumour cell migration may be a delay of at least one week, one month, several months, one year or longer.
  • the presence of metastases may be assessed using MRI, CT or Echography or techniques allowing the detection of circulating tumour cells (CTC). Examples of the latter tests are CellSearch CTC test (Veridex), an EpCam-based magnetic sorting of CTCs from peripheral blood.
  • tumour growth may be delayed or inhibited at least one day, two days, three days, four days, five days, six days or one week, two weeks, three weeks, one month, two months or more.
  • an occurrence of metastases is delayed at least one week, two weeks, three weeks, four weeks, one months, two months, three months, four months, five months, six months or more.
  • the population or composition according to the invention exerts its anti-cancer activity through the mechanism of radiotoxicity once it is bound to a cancer or tumour cell or metastatic lesion or CAF expressing the antigen, preferably human FAP or human FOLR1 or human HER2.
  • Tumour cell cancer cell
  • lesion metastatic lesion and dose of the combination have been defined in the section entitled definition.
  • a method for the prevention and/or treatment of a disease and/or disorder and/or condition comprising administering to a subject in need thereof, a population according to the invention or a composition according to the invention. All features of this method have been defined earlier herein.
  • Polypeptide nucleic acid, identity, similarity
  • amino acid residues will be indicated either by their full name or according to the standard three- letter or one-letter amino acid code.
  • polypeptide or ‘protein’ are used interchangeably, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • a “peptide” is also a polymer of amino acids with a length which is usually of up to 50 amino acids.
  • a polypeptide or peptide is represented by an amino acid sequence.
  • nucleic acid molecule As used herein, the terms ‘nucleic acid molecule’, ‘polynucleotide’, ‘polynucleic acid’, ‘nucleic acid’ are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof.
  • a nucleic acid molecule is represented by a nucleic acid sequence, which is primarily characterized by its base sequence.
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • the term 'homology' denotes at least secondary structural identity or similarity between two macromolecules, particularly between two polypeptides or polynucleotides, from same or different taxons, wherein said similarity is due to shared ancestry.
  • the term 'homologues' denotes so-related macromolecules having said secondary and optionally tertiary structural similarity.
  • Each amino acid sequence described herein by virtue of its identity percentage (at least 80%) with a given amino acid sequence respectively has in a further preferred embodiment an identity of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more identity with the given amino acid sequence respectively.
  • sequence identity is determined by comparing the whole length of the sequences as identified herein.
  • sequence similarity is determined by comparing the whole length of the sequences as identified herein. Unless otherwise indicated herein, identity or similarity with a given SEQ ID NO means identity or similarity based on the full length of said sequence (/.e. over its whole length or as a whole).
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences.
  • the identity between two amino acid sequences is preferably defined by assessing their identity within a whole SEQ ID NO as identified herein or part thereof. Part thereof may mean at least 50% of the length of the SEQ ID NO, or at least 60%, or at least 70%, or at least 80%, or at least 90%.
  • identity also means the degree of sequence relatedness between amino acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1 ): 387 (1984)), BestFit, FASTA, BLASTN, and BLASTP (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990)), EMBOSS Needle (Madeira, F., et al., Nucleic Acids Research 47(W1 ): W636-W641 (2019)).
  • the BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)).
  • the EMBOSS program is publicly available from EMBL-EBI. The well-known Smith Waterman algorithm may also be used to determine identity.
  • the EMBOSS Needle program is the preferred program used. Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48 (3):443-453 (1970); Comparison matrix: BLOSUM62 from Henikoff and Henikoff, Proc. Natl. Acad.
  • Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: DNAfull; Gap Open Penalty: 10; Gap Extend Penalty: 0.5.
  • a program useful with these parameters is publicly available as the EMBOSS Needle program from EMBL-EBI.
  • the aforementioned parameters are the default parameters for a Global Pairwise Sequence alignment of nucleotide sequences (along with no penalty for end gaps).
  • amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having acidic side chains is aspartate and glutamate; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys or Gin; Asn to Asp, His or Ser; Asp to Glu or Asn; Gin to Glu, Lys or Arg; Glu to Lys, Asp, Gin; His to Tyr or Asn; lie to Leu, Vai, or Met; Leu to lie, Met or Vai; Lys to Arg, Gin or Glu; Met to Vai, Leu or lie; Phe to Trp or Tyr; Ser to Thr, Ala or Asn; Thr to Ser; Trp to Tyr or Phe; Tyr to His, Trp or Phe; and Vai to lie, Leu or Met.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • an antibody refers to polyclonal antibodies, monoclonal antibodies, humanized antibodies, single-chain antibodies, and fragments thereof such as Fab F(ab')2, scFv, VHH and other fragments that retain the antigen binding function of the parent antibody.
  • an antibody may refer to an immunoglobulin or glycoprotein, or fragment or portion thereof, or to a construct comprising an antigen-binding portion comprised within a modified immunoglobulin-like framework, orto an antigen-binding portion comprised within a construct comprising a non-immunoglobulin-like framework or scaffold.
  • the term ‘monoclonal antibody’ refers to an antibody composition having a homogeneous antibody population.
  • the term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made.
  • the term encompasses whole immunoglobulins as well as fragments and others that retain the antigen binding function of the antibody.
  • Monoclonal antibodies of any mammalian species can be used in this invention. In practice, however, the antibodies will typically be of rat or murine origin because of the availability of rat or murine cell lines for use in making the required hybrid cell lines or hybridomas to produce monoclonal antibodies.
  • the term ‘polyclonal antibody’ refers to an antibody composition having a heterogeneous antibody population. Polyclonal antibodies are often derived from the pooled serum from immunized animals or from selected humans.
  • ‘Heavy chain variable domain of an antibody or a fragment thereof, as used herein, means (i) the variable domain of the heavy chain of a heavy chain antibody, which is naturally devoid of light chains (also indicated hereafter as VHH), including but not limited to the variable domain of the heavy chain of heavy-chain antibodies of camelids or sharks or (ii) the variable domain of the heavy chain of a conventional four-chain antibody (also indicated hereafter as VH), including but not limited to a camelized (as further defined herein) variable domain of the heavy chain of a conventional four-chain antibody (also indicated hereafter as camelized VH) or any fragments thereof, such as but not limited to one or more stretches of amino acid residues (i.e.
  • the fragment of a VHH is a functional fragment.
  • the amino acid sequence and structure of a heavy chain variable domain of an antibody can be considered, without however being limited thereto, to be comprised of four framework regions or ‘PR's’, which are referred to in the art and herein below as ‘framework region T or ‘FR1 as ‘framework region 2’ or ‘FR2’; as ‘framework region 3’ or ‘FR3’; and as ‘framework region 4’ or ‘ FR4’ , respectively, which framework regions are interrupted by three complementary determining regions or ‘CDR's’, which are referred to in the art as ‘complementarity determining region T or ‘CDR1 ’; as ‘complementarity determining region 2’ or ‘CDR2’; and as ‘complementarity determining region 3’ or ‘CDR3’, respectively.
  • CDR's complementary determining regions
  • variable regions of either the H (heavy) or the L (light) chains (also abbreviated as VH and VL, respectively) and contain the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions.”
  • the CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains.
  • the variable heavy and light chains of all canonical antibodies each have 3 CDR regions, each non- contiguous with the others (termed L1 , L2, L3, H1 , H2, H3) for the respective light (L) and heavy (H) chains.
  • the total number of amino acid residues in a heavy chain variable domain of an antibody can be in the region of 110-130.
  • parts, fragments or analogs of a heavy chain variable domain of an antibody are not particularly limited as to their length and/or size, as long as such parts, fragments or analogs retain (at least part of) the functional activity, and/or retain (at least part of) the binding specificity of the original heavy chain variable domain of an antibody from which these parts, fragments or analogs are derived from.
  • Parts, fragments or analogs retaining (at least part of) the functional activity, and/or retaining (at least part of) the binding specificity of the original heavy chain variable domain of an antibody from which these parts, fragments or analogs are derived from are also further referred to herein as ‘functional fragments’ of a heavy chain variable domain.
  • variable domains of an antibody are preferably numbered according to the IMGT unique numbering for V-domain (immunoglobulins and T cell receptors) given by the IMGT nomenclature as described (Lefranc M.P. et al 1997 Immunology today, 18: 509, PMID: 9386342; Lefranc, M.-P., 1999 The Immunologist, 7: 132-136 and Lefranc M.P. et al 2003, Dev. Comp. Immunol., 27: 55-77 PMID: 12477501 ).
  • the conserved amino acids always have the same position, for instance cysteine 23 (1 st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine 104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP).
  • the IMGT unique numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 1 18 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. Gaps represent unoccupied positions. Gaps in the CDR1-IMGT and CDR2-IMGT (less than 12 and 10 amino acid long, respectively) are put at the top of the CDR-IMGT loops.
  • the basic length of a rearranged CDR3-IMGT is 13 amino acids (positions 105 to 117), which corresponds to a JUNCTION of 15 amino acids (2nd-CYS 104 to J-TRP or J-PHE 1 18). If the CDR3- IMGT length is less than 13 amino acids, gaps are created from the top of the loop, in the following order 111 , 112, 110, 113, 109, 114, etc. If the CDR3-IMGT length is more than 13 amino acids, additional positions are created between positions 111 and 112 at the top of the CDR3-IMGT loop in the following order 112.1 ,111.1 , 112.2, 111.2, 112.3, 111.3, etc.
  • the total number of amino acid residues in each of the CDR's may vary and may not correspond to the total number of amino acid residues indicated by the IMGT numbering (that is, one or more positions according to the IMGT numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the IMGT numbering).
  • the numbering according to IMGT may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • amino acid residues of a variable domain of an antibody can be numbered according to Kabat numbering (Kabat et al 1987, National Institute of Health; 1987. 804 pp., Publication no. 165-462.). Correspondence between the IMGT and Kabat numbering for the immunoglobulin V-regions can be found for example in Table 2 of Lefranc et al., 2003.
  • heavy chain variable domain as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation.
  • heavy chain variable domains derived from heavy chain antibodies i.e.
  • VHH’s can be obtained (1 ) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by ‘camelization’ (as described below) of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (4) by ‘camelization’ of a ‘domain antibody’ or ‘dAb’ as described by Weizao C., et al Methods Mol Biol 2009, 525:81-99)), or by expression of a nucleic acid encoding such a camelized VH domain (5) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (6) by preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus
  • An antibody fragment such as a single-domain antibody fragment as disclosed herein is considered to be '(in) essentially isolated (form)' as used herein, when it has been extracted or purified from the host cell and/or medium in which it is produced.
  • antibody fragments are obtained by affinity selection an antigen present on and/or specific for a solid tumour and/or a cancer cell, preferably human and/or murine FAP, or human FOLR1 .
  • Obtaining suitable polypeptides by affinity selection against a particular solid tumour antigen or cancer cell may for example be performed by screening a set, collection or library of cells that express antibody fragments on their surface (e.g.
  • bacteriophages for binding against a tumour-specific antigen and/or a cancer cell-specific antigen; all of which may be performed in a manner known per se, essentially comprising the following non-limiting steps: a) obtaining an isolated solution or suspension of a tumour-specific or cancer cell-specific protein target molecule, which molecule is known to be a target for a potential cancer drug; b) bio-panning phages or other cells from a antibody fragment library against said protein target molecule; c) isolating the phages or other cells binding to the tumour-specific or cancer cell-specific protein target molecule; d) determining the nucleotide sequence encoding the antibody fragment insert from individual binding phages or other cells; e) producing an amount of antibody fragment according to this sequence using recombinant protein expression; f) determining the affinity of said antibody fragment domain for said tumour-specific or cancer cell-specific protein target molecule; and optionally g) testing the tumouricidal or anticancer activity of said antibody fragment
  • Various methods may be used to determine the affinity between the antibody fragment domain and the tumour-specific or cancer cell-specific protein target molecule, including for example, enzyme linked immunosorbent assays (ELISA) or Surface Plasmon Resonance (SPR) assays, which are common practice in the art, for example, as described in Sambrook et al. (2001 ), Molecular Cloning, A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • the equilibrium dissociation constant is commonly used to describe the affinity between a polypeptide and its target molecule.
  • the equilibrium dissociation constant is lower than 10' 7 M.
  • the equilibrium dissociation constant is lower than 10' 8 M, or lower than 10' 9 M, or more preferably, ranged from 10 ® M to 10' 12 M.
  • the antibody fragments are not limited as to the origin of the antibody fragments (or of the nucleotide sequences of the invention used to express them). Furthermore, the present invention is also not limited as to the way that the antibody fragments have been generated or obtained.
  • the amino acid sequences as disclosed herein may be synthetic or semi-synthetic amino acid sequences, polypeptides or proteins.
  • the present invention also encompasses parts, fragments, analogues, mutants, variants, and/or derivatives of the antibody fragments and/or polypeptides comprising or essentially consisting of one or more of such parts, fragments, analogues, mutants, variants, and/or derivatives, as long as these parts, fragments, analogues, mutants, variants, and/or derivatives are suitable for the purposes envisaged herein, which are preferably: deliver to a CNS molecule, a cell, a tissue or an organ expressing an antigen, preferably FAP or FOLR1 , linked to it and suitable to be used in diagnostic and therapeutic applications when linked to a radionuclide.
  • Such parts, fragments, analogues, mutants, variants, and/or derivatives according to the invention are preferably still capable of specifically binding said antigen, preferably to:
  • - are preferably not modulators of FAP, preferably not inhibitors of FAP, and/or - specifically bind to human FOLR1 and not capable of specifically binding to murine FOLR1 and to human FOLR2 and to human FOLR3.
  • a number of stretches of amino acid residues are provided herein, also referred to CDR sequences or part of the antibody fragment and identified as SEQ ID NO: 1 , 2, 3, such as sequences having at least 80% identity with SEQ ID NO: 4 representing the sequence of the antibody fragment that are particularly suited for binding to human and/or murine FAP.
  • a number of stretches of amino acid residues are provided herein, also referred to herein as CDR sequences or part of the antibody fragment and identified as SEQ ID NO, such as sequences having at least 60% identity with at least one of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 31 , 32 of the antibody fragment that are particularly suited for binding to human FOLR1.
  • These stretches may be regarded as being functional fragments of the antibody fragment and may be present in, and/or may be incorporated into any suitable scaffold (protein), in particular in such a way that they form (part of) the antigen binding site of that suitable scaffold or antibody fragment. It should however be noted that the invention in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in the scaffolds or antibody fragments as disclosed herein, as long as these stretches of amino acid residues allow these scaffolds or antibody fragments as disclosed herein to specifically bind to said antigen.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be optionally linked to one or more further groups, moieties, or residues via one or more linkers. These one or more further groups, moieties or residues can serve for binding to other targets of interest. It should be clear that such further groups, residues, moieties and/or binding sites may or may not provide further functionality to the antibody fragments and may or may not modify its properties as disclosed herein. Such groups, residues, moieties or binding units may also for example be chemical groups which can be biologically active.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may also have been chemically modified.
  • a modification may involve the introduction or linkage of one or more functional groups, residues or moieties into or onto the antibody fragment. These groups, residues or moieties may confer one or more desired properties or functionalities to the antibody fragment. Examples of such functional groups will be clear to the skilled person.
  • the introduction or linkage of such functional groups to the antibody fragment can result in an increase in their solubility and/or their stability, in a reduction of their toxicity, or in the elimination or attenuation of any undesirable side effects, and/or in other advantageous properties.
  • one or more groups, residues or moieties are linked to an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof via one or more suitable linkers or spacers.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof are directed against or specifically bind to the same site, determinant, part, epitope, domain or stretch of amino acid residues of an antigen as disclosed herein is said to be ‘bivalent’ (in the case of two binding sites on the antibody fragment) or multivalent (in the case of more than two binding sites on the antibody fragment), such as for example trivalent.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is present in a monovalent format.
  • the term ‘monovalent’ when referring to an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof denotes an antibody fragment in monomeric form.
  • a monovalent antibody fragment contains only one binding site.
  • the binding site of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof encompasses one or more ‘complementarity determining regions’ or ‘CDRs’ that are directed against or specifically bind to a particular site, determinant, part, epitope, domain or stretch of an antigen.
  • the binding site of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof encompasses one or more ‘complementarity determining regions’ or ‘CDRs’ represented by SEQ ID NO:1 , 2 and/or 3 and/or one or more regions identified herein as having at least 80% identity with SEQ ID NO: 4 of an antibody fragment that is directed against or specifically bind to a particular site, determinant, part, epitope, domain or stretch of amino acid residues of human and/or murine FAP.
  • the binding site of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof encompasses the one or more ‘complementarity determining regions’ or ‘CDRs’ and/or the one or more regions identified herein as having at least 60% identity with at least one of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 31 , 32 of a an antibody fragment that is directed against or specifically bind to a particular site, determinant, part, epitope, domain or stretch of amino acid residues of human FOLR1.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is attractive for therapeutic/diagnostic applications.
  • a small size may also be attractive if a high tissue penetration is needed in order to reach an optimal therapeutic effect.
  • linkers or spacers include for example, but are not limited to, polypeptide linkers such as glycine linkers, serine linkers, mixed glycine/serine linkers, glycine- and serine-rich linkers or linkers composed of largely polar polypeptide fragments, or homo- or heterobifunctional chemical crosslinking compounds such as glutaraldehyde or, optionally PEG-spaced, maleimides or NHS esters.
  • polypeptide linkers such as glycine linkers, serine linkers, mixed glycine/serine linkers, glycine- and serine-rich linkers or linkers composed of largely polar polypeptide fragments, or homo- or heterobifunctional chemical crosslinking compounds such as glutaraldehyde or, optionally PEG-spaced, maleimides or NHS esters.
  • a polypeptide linker or spacer may be a suitable amino acid sequence having a length from 1 to 50 amino acids, such as from 1 to 30, and in particular from 1 to 10 amino acid residues. It should be clear that the length, the degree of flexibility and/or other properties of the linker(s) may have some influence on the properties of the antibody fragments, including but not limited to the affinity, specificity or avidity for the tumour target or the target on a cancer cell or pharmacological behaviour. It should be clear that when two or more linkers are used, these linkers may be the same or different.
  • the term ‘untagged’ when referring to an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof denotes an antibody fragment that contains no extraneous polypeptide sequences (e.g., contains only said antibody fragment sequence).
  • exemplary extraneous polypeptide sequences include carboxy-terminal polypeptide tags, e.g., a His-tag, a cysteine-containing tag (e.g., a GGC-tag as described in Pruszynski et al 2013 Nucl Med Biol 40: 52-59), and/or a Myc-tag.
  • a His-tag may contain 4, 5, 6, 7, 8, 9, 10 Histidines. In an embodiment, 6 Histidines are present.
  • the one or more groups, residues or moieties that may be present do not induce multimerization such as dimerization of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof. Therefore in an embodiment, an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is devoid of a tag that induces multimerization such as dimerization, preferably devoid of a cysteine-containing tag, preferably a GGC-tag.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is devoid of a carboxy-terminal polypeptide tag, preferably it is untagged.
  • kidney retention was shown to be significantly reduced when using an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof without a carboxy-terminal polypeptide tag compared to a polypeptide tagged, such as His-tagged and Myc-His-tagged antibody fragment (D'Huyvetter et al. (2014), Theranostics. 4(7):708-20).
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof without a carboxy-terminal polypeptide tag compared to a polypeptide tagged, such as His-tagged and Myc-His-tagged antibody fragment (D'Huyvetter et al. (2014), Theranostics. 4(7):708-20).
  • bi-specific when referring to an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof implies that either a) two or more of the binding sites of the antibody fragment are directed against or specifically bind an antigen but not to the same (i.e.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is said to be ‘bi-specific’ (in the case of two binding sites on the antibody fragment) or multispecific (in the case of more than two binding sites on antibody fragment) or b) two or more binding sites of the antibody fragment are directed against or specifically bind to different target molecules of interest.
  • the term ‘multispecific’ is used in the case that more than two binding sites are present on the antibody fragment.
  • the 'half-life' of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof can generally be defined as the time that is needed for the in vivo serum concentration of the antibody fragment to be reduced by 50%.
  • the in vivo half-life of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof can be determined in any manner known to the person skilled in the art, such as by pharmacokinetic analysis. As will be clear to the skilled person, the half-life can be expressed using parameters such as the t1 /2-alpha, t1/2-beta and the area under the curve (AUC).
  • An increased half-life in vivo is generally characterized by an increase in one or more and preferably in all three of the parameters tiealpha, t1/2-beta and the area under the curve (AUC).
  • the term “lifetime extended” when referring to an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is used to denote that the antibody fragment has been modified to extend the half-life of the antibody fragment.
  • Strategies for extending the half-life of antibody fragments are well-known in the art and include for example, but without limitation, linkage (chemically or otherwise) to one or more groups or moieties that extend the half-life, such as polyethylene glycol (PEG) or bovine serum albumin (BSA) or human serum albumin (HSA), antibody Fc fragments, or antigen-binding antibody fragments targeting serum proteins such as serum albumin.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is non-lifetime extended.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof can be determined in any suitable manner known per se, including for example biopanning, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) (also called Enzyme-Linked Immuno Sorbent Assay, ELISA), sandwich competition assays, Surface Plasmon Resonance (SPR), or Bio-Layer Interferometry and the different variants thereof known in the art.
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • ELISA Enzyme-Linked Immuno Sorbent Assay
  • sandwich competition assays such as sandwich competition assays, Surface Plasmon Resonance (SPR), or Bio-Layer Interferometry and the different variants thereof known in the art.
  • SPR Surface Plasmon Resonance
  • Each of these assays may be carried out in vitro using an antigen, preferably a human and/or murine FAP recombinant protein or a human FOLR1 recombinant protein or a human HER2 recombinant protein, which may be immobilised on a support or in solution.
  • some of these assays may be carried out in vitro using cells that express the corresponding antigen, preferably human and/or murine FAP, or human FOLR1 , or human HER2.
  • Such cells may endogenously express or overexpress express the antigen, preferably human and/or murine FAP, or human FOLR1 , or human HER2.
  • the assessment is usually carried out in vitro in a culture medium or in PBS or in a suitable medium or buffer.
  • a preferred cell is a fibroblast cell expressing human and/or murine FAP.
  • a preferred cell line may be GM05389 or U-87 MG.
  • a preferred cell is a transfected cell expressing human and/or murine FAP.
  • a preferred transfected cell line may be HEK293.
  • a preferred cell is SKOV3 or OVCAR3 expressing human FOLR1.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be assessed in vivo in an animal expressing the corresponding antigen, preferably human and/or murine FAP or human FOLR1 or human HER2, preferably using an imaging technique is used.
  • a preferred imaging technique is SPECT/CT, PET/CT, SPECT/MRI or PET/MRI.
  • Cells overexpressing an antigen, preferably human and/or murine FAP or human FOLR1 or human HER2 may also be xenografted into the animal.
  • in vitro is therefore used herein in the context of a cell-free assay when the human and/or murine FAP or human FOLR or human HER2 recombinant protein is immobilized on a support or in solution, or in the context of a cell in culture.
  • in vivo or “ex vivo” is used herein in the context of a non-human animal or a tissue or organ of this non-human animal.
  • ex vivo is used when a quantification is carried out on a tissue or organ of a non-human or human animal and “in vivo” is used when a quantification via an imaging method is carried out on a non-human or human animal.
  • binding is assessed using in vitro conditions and is further confirmed using in vivo conditions.
  • affinity refers to the degree to which an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof binds to an antigen, preferably human and/or murine FAP or human FOLR1 or human HER2, so as to shift the equilibrium of the antibody fragment and the antigen towards the presence of a complex formed by their binding.
  • the binding may be assessed using SPR or bio-layer interferometry.
  • the antibody fragment of high affinity will bind to the available human and/or murine FAP so as to shift the equilibrium towards high concentrations of the resulting complex.
  • the equilibrium dissociation constant (KD) is commonly used to describe the affinity between the protein binding domain (antibody fragment) and the antigen.
  • the equilibrium dissociation constant is less than 10' 7 M.
  • the equilibrium dissociation constant is less than 10' 8 M, or less than 10' 9 M, or more preferably, ranging from 10' 9 M and 10' 12 M.
  • Any antibody fragment as disclosed herein is preferably such that it specifically binds (as defined herein) to human and/or murine FAP or human FOLR1 or human HER2 with an equilibrium dissociation constant (KD) ranging from 10' 9 to 10' 12 moles/liter or from 10' 1 ° to 10' 12 moles/liter, preferably assessed using bio-layer interferometry.
  • KD equilibrium dissociation constant
  • the 'specificity' of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof can be determined based on affinity and/or avidity.
  • the 'affinity' of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is represented by the equilibrium constant for the dissociation of the antibody fragment and the antigen to which it binds. The lower the KD value, the stronger the binding strength between the antibody fragment and the target protein (antigen) of interest to which it binds.
  • the affinity can also be expressed in terms of the equilibrium association constant (KA), which corresponds to 1/KD.
  • the binding affinity of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof can be determined in a manner known to the skilled person, depending on the specific target protein of interest.
  • the 'avidity' of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is the measure of the strength of binding between the antibody fragment and the pertinent target protein of interest.
  • Avidity is related to both the affinity between a binding site on the target protein of interest and a binding site on the antibody fragment and the number of pertinent binding sites present on the antibody fragment.
  • VHHs have only one single-domain and therefore only one single binding site.
  • the affinity exhibited by VHHs is in the sub-nanomolar range and is therefore quite exceptional in view of the presence of a single binding site.
  • a KD value greater than about 1 millimolar is generally considered to indicate nonbinding or non-specific binding. It is generally known in the art that the KD can also be expressed as the ratio of the dissociation rate constant of a complex, denoted as k O ff or kd (expressed in seconds 1 or s -1 ), to the rate constant of its association, denoted k on or k a (expressed in molar 1 seconds 1 or M' 1 s -1 ).
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may bind to the target protein of interest (i.e. antigen) with a kon ranging from 0.1 and 0.00001 s -1 , or ranging from 10' 2 to 10' 5 s -1 , or ranging from 10' 3 to 10' 5 s' 1 , or ranging from 10' 4 to 10' 5 s 1 and/or a k on ranging from 1 ,000 and 10,000,000 M' 1 s 1 or ranging from 10 4 to 10 7 M' 1 s' 1 or from 10 5 to 10 7 M' 1 s' 1 .
  • Binding affinities, k O ff and k on rates may be determined by means of methods known to the person skilled in the art, for example ELISA methods, isothermal titration calorimetry, SPR, bio-layer interferometry, fluorescence-activated cell sorting analysis, and the more.
  • the antibody fragment as disclosed herein specifically binds to human and/or murine FAP with a k O ff ranging from 0.1 and 0.00001 s 1 , or ranging from 10' 2 to 10' 5 s 1 or from 10' 3 to 10' 5 s 1 , or ranging from 10' 4 to 10' 5 s 1 preferably assessed using bio-layer interferometry.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof as disclosed herein is such that it specifically binds (as defined herein) to human and/or murine FAP with a KD ranged from 10' 9 to 10' 12 moles/liter and/or a k O ff ranging from 10' 2 to 10' 5 s 1 preferably assessed using bio-layer interferometry, more preferably with a KD ranged from 10' 9 to 10' 12 moles/liter and a k O ff ranging from 10' 2 to 10' 5 s' 1
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof is said to ‘specifically bind to’ an antigen, preferably human and/or murine FAP or human FOLR1 or human HER2, when that an antibody fragment thereof has affinity for, specificity for and/or is specifically directed against that antigen (or for at least one part or fragment thereof).
  • an antigen preferably human and/or murine FAP or human FOLR1 or human HER2
  • the terms 'binding region', 'binding site' or 'interaction site' present on the antibody fragment shall herein have the meaning of a particular site, part, locus, domain or stretch of amino acid residues present on the antibody fragment that is responsible for binding or specific binding to the corresponding antigen, preferably human and/or murine FAP or human FOLR1 or human HER2.
  • This binding region present on the antibody fragment is called a paratope.
  • Such binding region comprises, consists or essentially consists of specific amino acid residues from the amino acid sequence as disclosed herein of the antibody fragment which are in contact with the corresponding antigen, preferably with human and/or murine FAP or human FOLR1 or human HER2.
  • the region or part or discrete amino acids of the extracellular domain of the antigen, preferably human and/or murine FAP or human FOLR1 or human HER2, that is in contact with said antibody fragment may be called an epitope and are defined later herein.
  • An antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof may be produced or manufactured by any of the methods describes below.
  • one particularly useful method for preparing an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof generally comprises the steps of:
  • the nucleic acid encoding antibody fragment may be comprised in a vector or genetic construct.
  • the antibody fragment can be obtained by methods which involve generating a random library of antibody fragment sequences and screening this library for a antibody fragment sequence capable of specifically binding to a corresponding antigen, preferably human and/or murine FAP or human FOLR1 or human HER2.
  • the set, collection or library of antibody fragment sequences may be any suitable set, collection or library of amino acid sequences.
  • the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin fragment sequences (as described herein), such as a naive set, collection or library of immunoglobulin fragment sequences; a synthetic or semi-synthetic set, collection or library of immunoglobulin fragment sequences; and/or a set, collection or library of immunoglobulin fragment sequences that have been subjected to affinity maturation.
  • the set, collection or library of antibody fragment sequences may be an immune set, collection or library of immunoglobulin fragment sequences, for example derived from a mammal that has been suitably immunized with a corresponding antigen, preferably human and/or murine FAP or human FOLR1 or human HER2 or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof.
  • said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).
  • the set, collection or library of antibody fragment sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening.
  • suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).
  • the collection or sample of cells may for example be a collection or sample of B-cells.
  • the sample of cells may be derived from a mammal that has been suitably immunized with a corresponding antigen, preferably human and/or murine FAP or human FOLR1 or human HER2, or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof.
  • the antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).
  • the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naive set, collection or library of immunoglobulin fragment sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin fragment sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin fragment sequences that have been subjected to affinity maturation.
  • the set, collection or library of nucleic acid sequences encodes a set, collection or library of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof directed against an antigen, preferably human and/or murine FAP or human FOLR1 or human HER2.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof directed against an antigen, preferably human and/or murine FAP or human FOLR1 or human HER2.
  • the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening.
  • suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 110S- 1116 (2005).
  • the invention also relates to an antibody fragment such as a single-domain antibody, preferably a VHH, or a fragment thereof that are obtainable or obtained by the above methods, or alternatively by a method that comprises one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said antibody fragment; and of expressing or synthesizing said single-domain antibody in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.
  • an antibody fragment such as a single-domain antibody, preferably a VHH, or a fragment thereof that are obtainable or obtained by the above methods, or alternatively by a method that comprises one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said antibody fragment; and of expressing or synthesizing said single-domain antibody in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.
  • the methods for producing an antibody fragment such as a single-domain antibody, preferably a VHH, or a fragment thereof binding specifically to an antigen, preferably to human and/or murine FAP or human FOLR1 or human HER2, may further comprise the step of isolating from the amino acid sequence library at least one antibody fragment having detectable binding affinity for, or detectable in vitro effect on the antigen.
  • These methods may further comprise the step of amplifying a sequence encoding at least one antibody fragment thereof having detectable binding affinity for, or detectable in vitro effect on the activity of the antigen, preferably of human and/or murine FAP or human FOLR1 or human HER2.
  • a phage clone displaying a particular amino acid sequence obtained from a selection step of a method described herein, may be amplified by reinfection of a host bacteria and incubation in a growth medium.
  • Particular methods may encompass determining the sequence of the one or more amino acid sequences capable of binding to the antigen, preferably human and/or murine FAP or human FOLR1 or human HER2.
  • an antibody fragment such as a single-domain antibody, preferably a VHH, or a fragment thereof, comprised in a set, collection or library of amino acid sequences, is displayed on a suitable cell or phage or particle, it is possible to isolate from said cell or phage or particle, the nucleotide sequence that encodes that amino acid sequence. In this way, the nucleotide sequence of the selected amino acid sequence library member(s) can be determined by a routine sequencing method.
  • the obtained antibody fragment having detectable binding affinity for, and/or no detectable in vitro effect on an activity of the antigen may be synthesized as soluble protein construct, optionally after their sequence has been identified.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof obtained, obtainable or selected by the above methods can be synthesized using recombinant or chemical synthesis methods known in the art.
  • the amino acid sequences obtained, obtainable or selected by the above methods can be produced by genetic engineering techniques.
  • methods for synthesizing the antibody fragment obtained, obtainable or selected by the above methods may comprise transforming or infecting a host cell with a nucleic acid or a vector encoding an amino acid sequence having detectable binding affinity for, and/or no detectable in vitro effect on an activity of the antigen, preferably human and/or murine FAP or human FOLR1 or human HER2.
  • the antibody fragment having detectable binding affinity for, and/or no detectable in vitro effect on an activity of the antigen can be made by recombinant DNA methods.
  • DNA encoding the amino acid sequences can be readily synthesized using conventional procedures. Once prepared, the DNA can be introduced into expression vectors, which can then be transformed or transfected into host cells such as E. coli or any suitable expression system, in order to obtain the expression of amino acid sequences in the recombinant host cells and/or in the medium in which these recombinant host cells reside.
  • an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof produced from an expression vector using a suitable expression system may be tagged (typically at the N-terminal or C- terminal end of the amino acid sequence) with e.g. a His-tag or other sequence tag for easy purification.
  • Transformation or transfection of nucleic acids or vectors into host cells may be accomplished by a variety of means known to the person skilled in the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • Suitable host cells for the expression of the desired heavy chain variable domain sequences may be any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
  • host cells may be located in a transgenic plant.
  • the application also provides methods for the production of an antibody fragment such as a single-domain antibody fragment, preferably a VHH, or a fragment thereof having detectable binding affinity for, or detectable in vitro effect on the activity of an antigen, preferably human and/or murine FAP or human FOLR1 or human HER2, comprising transforming, transfecting or infecting a host cell with nucleic acid sequences or vectors encoding such antibody fragment and expressing their amino acid sequences under suitable conditions.
  • an antigen preferably human and/or murine FAP or human FOLR1 or human HER2
  • the dose is defined as the amount of label administered due to the administration of said population or composition.
  • the administration of given dose of a label refers to the use of a population according to the invention or a composition according to the invention, wherein the amount of the label during the administration is said dose.
  • a dose refers to applications wherein the label comprises a radionuclide.
  • DLT dose-limiting toxicity
  • MTD maximum tolerated dose
  • the use of a population according to the invention or a composition according to the invention comprises the administration of radionuclide (i.e. label) at a radioactive dosage of lower than about 800 mCi, such as for instance lower than about 150 mCi, such as for instance lower than about 30 mCi, such as lower than about 15 mCi.
  • radionuclide i.e. label
  • the use of a population according to the invention or a composition according to the invention comprises the administration of a radionuclide with a specific activity from about 0.5 mCi/mg to about 8000 mCi/mg, such as for instance from 1 mCi/mg to about 1500 mCi/mg, such as for instance from 1 mCi/mg to about 300 mCi/mg, such as for instance from 1 mCi/mg to about 150mCi/mg, depending on the radionuclide.
  • the first and/or administration may be intravenous, intraperitoneal or via another route such as intrathecal.
  • the population or composition may be administered once or several times, in combination with other therapeutic drugs or radio-sensitizing agents.
  • the amount of the label applied depends on the precise nature of the carcinoma.
  • the dose of radioactivity per administration must be high enough to be effective, but must be below the dose limiting toxicity (DLT).
  • a “screening dose” or a “biomarker dose” is a dose of a label as described herein, that is sufficient for selecting a subject for treatment, such as a dose that can bind to a cancer cell or solid tumour in the subject and subsequently be detected at the location of the cancer cell or solid tumour, e.g., by imaging the subject using gamma camera imaging such as planar gamma camera imaging, single photon emission computed tomography or positron emission tomography, optionally combined with a non-nuclear imaging technique such as X-ray imaging, computed tomography and/or magnetic resonance imaging.
  • a screening dose is a dose that is not therapeutically effective.
  • the screening dose is different than (e.g., lower than) a therapeutic dose as described herein.
  • a “therapeutic dose” is a dose of a label as described herein, that is therapeutically effective in at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of subjects in need of such treatment (e.g., in subjects having cancer).
  • the therapeutic dose is higher than a screening dose as described herein.
  • imaging a subject refers to capturing one or more images of a subject using a device that is capable of detecting a label as described herein.
  • the one or more images may be further altered by a computer program and/or a person skilled in the art in order to enhance the images (e.g. by adjusting contrast or brightness of the one or more images).
  • Any device capable of detecting label as described herein is contemplated for use, such as a device for gamma camera imaging such as planar gamma camera imaging, for single photon emission computed tomography or for positron emission tomography, or a device able to combine a nuclear imaging technique with an anatomical imaging technique such as X-ray imaging, computed tomography and/or magnetic resonance imaging.
  • such device can be a device for single photon emission computed tomography/computed tomography (SPECT/CT) or positron emission computed tomography/computed tomography (PET/CT) imaging.
  • SPECT/CT single photon emission computed tomography/computed tomography
  • PET/CT positron emission computed tomography/computed tomography
  • the administration of the screening dose and the detection by imaging are separated by at least 1 about minute, at least 5 about minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 1 .5 hours, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 7 days.
  • the administration of the screening dose and the detection are separated by between about 1 hour and about 24 hours.
  • the screening dose and the therapeutic dose are administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least one month, at least about 2 months, or at least about 6 months apart. In some embodiments, the screening dose and the therapeutic dose are administered between about 1 day and about 6 months apart (e.g., between about 1 day and about 2 months, between about 1 day and about 1 month, or between about 1 day and about 1 week apart).
  • the screening dose and therapeutic dose may each independently be administered by any suitable route, such as systemically, locally or topically.
  • routes include intravenous, intraperitoneal, and intrathecal administration.
  • the particular route utilized may, in some embodiments, depend on the nature of the disease (e.g., type, grade, location and stage of the tumour or cancer cell etc.) and the type of subject (e.g., species, constitution, age, gender, weight, etc.).
  • the term “subject” generally refers to a mammal, such as a human, a non-human primate, a rat, a mouse, a rabbit, a dog, a cat, a pig, a horse, a goat, or a sheep.
  • the subject is a human subject.
  • the subject is a subject having cancer (e.g., a human subject having cancer).
  • Methods for identifying subjects having cancer include detection of tumour antigens or other tumour biomarkers, genetic testing, MRI, X-ray, PET or SPECT scan, biopsies, and combinations thereof.
  • the terms 'diagnosis’, ‘prediction’ and/or ‘prognosis' as used herein comprise diagnosing, predicting and/or prognosing a certain disease and/or disorder and/or condition, thereby predicting the onset and/or presence of a certain disease and/or disorder and/or condition, and/or predicting the progress and/or duration of a certain disease and/or disorder and/or condition, and/or predicting the response of a patient suffering from of a certain disease and/or disorder and/or condition to therapy.
  • a screening dose (i.e. used in a diagnostic method) is a dose that is not therapeutically effective.
  • the screening dose is lower than a therapeutic dose as described herein (e.g., at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times or at least about 1000 times lower than a therapeutic dose as described herein, or at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about
  • the screening dose is from about 10 MBq to about 400 MBq, from about 20 MBq to about 400 MBq, from about 30 MBq to about 400 MBq, from about 40 MBq to about 400 MBq, from about 50 MBq to about 400 MBq, from about 100 MBq to about 400 MBq, from about 200 MBq to about 400 MBq, from about 300 MBq to about 400 MBq, from about 10 MBq to about 300 MBq, from about 20 MBq to about 300 MBq, from about 30 MBq to about 300 MBq, from about 40 MBq to about 300 MBq, from about 50 MBq to about 300 MBq, from about 100 MBq to about 300 MBq, or from about 200 MBq to about 300 MBq.
  • the screening dose is from about 7 MBq to about 370 MBq. It is to be understood that any screening dose described herein may be combined with any therapeutic dose as described herein.
  • the therapeutic dose is higher than a screening dose as described herein (e.g., at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times or at least about 1000 times higher than a screening dose as described herein, or at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at
  • the therapeutic dose is from about 300 MBq to about 20000 MBq, from about 400 MBq to about 20000 MBq, from about 500 MBq to about 20000 MBq, from about 1000 MBq to about 20000 MBq, from about 2000 MBq to about 20000 MBq, from about 3000 MBq to about 20000 MBq, from about 4000 MBq to about 20000 MBq, from about 5000 MBq to about 20000 MBq, from about 10000 MBq to about 20000 MBq, from about 5000 MBq to about 20000 MBq, from about 10000 MBq to about 20000 MBq, from about 300 MBq to about 10000 MBq, from about 400 MBq to about 10000 MBq, from about 500 MBq to about 10000 MBq, from about 1000 MBq to about 10000 MBq, from about 2000 MBq to about 10000MBq, from about 3000 MBq to about 10000 MBq, from about
  • the screening and/or therapeutic dose may conveniently be presented in a single dose or as divided doses (which can again be sub-dosed) administered at appropriate intervals.
  • An administration regimen of the therapeutic dose could include long-term (e.g., at least two weeks, and for example several months or years) or daily treatment.
  • an administration regimen of the therapeutic dose can vary between once a day to once a month, such as between once a day and once every two weeks, such as but not limited to once a week.
  • combinations as disclosed herein may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months.
  • the particular screening dose and therapeutic dose utilized may, in some embodiments, depend on the nature of the disease (such as cancer but also the type, grade, and stage of the tumour or cancer cell etc., or such as any of the other diseases identified herein) and the type of subject (e.g., species, constitution, age, gender, weight, etc.).
  • the invention provides kits.
  • the kit comprises a screening dose of a label in the form of a population or composition according to the invention and a therapeutic dose of the same label in the form of a population or composition according to the invention. Screening doses and therapeutic doses are described herein.
  • the kit further comprises one or more means for injection of the screening dose and the therapeutic dose.
  • the screening dose and therapeutic dose are each individually housed in a means for injection.
  • the means for injection is a syringe.
  • the kit further comprises instructions for carrying out a method as described herein (e. g.
  • the instructions may be in any suitable form, e.g., in printed form (e g., as a paper or laminated insert or label) or in electronic form (e g., on a disc or USB stick).
  • Dose, route of administration, application scheme, repetition and duration of treatment will in general depend on the nature of the disease (type, grade, and stage of the tumour or cancer cell or type, grade and stage of the disease or condition further defined herein) and the patient (constitution, age, gender etc.), and will be determined by the skilled medical expert responsible for the treatment.
  • a composition according to the invention preferably for use as a medicament, comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds.
  • Such compositions may be suitable for intraperitoneal, intravenous or other administration such as intrathecal administration.
  • the administration may be systemically, locally or topically to the tissue or organ of interest, depending on the location, type and origin of the tumour or cancer cell, and preferably intraperitoneally, intravenously or intrathecally, depending on the specific pharmaceutical formulations or compositions to be used.
  • the clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredients which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the amount of the label as envisaged herein required for use in prophylaxis and/or treatment may vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also, the dosage of the label envisaged herein may vary depending on the target cell, tumour, tissue, graft, or organ.
  • the label as envisaged herein will be administered in an amount which will be determined by the medical practitioner based inter alia on the severity of the condition and the patient to be treated.
  • an optimal dosage will be determined specifying the amount to be administered per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day.
  • the clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment.
  • Useful dosages of the label as envisaged herein can be determined by determining their in vitro activity, and/or in vivo activity in animal models.
  • the populations or combinations according to the invention are for use in the prevention and/or treatment of cancer (preferably cancer which is associated with the expression of human FAP on cancer cells and/or on CAF) by administration from 10 pg and 10 mg or from 10 pg and 7 mg or from 10 pg and 5 mg or from 10 pg and 2 mg or from 10 pg and 1.5 mg or from 10 pg and 1 mg of antibody fragment.
  • the dose of radioactivity applied to the patient per administration has to be high enough to be effective but must be below the dose limiting toxicity (DLT).
  • DLT dose limiting toxicity
  • the maximally tolerated dose (MTD) has to be determined which must not be exceeded in therapeutic settings.
  • the administration is preferably according to a regimen of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated.
  • the clinician will generally be able to determine a suitable treatment regimen.
  • the treatment regimen will comprise the administration of a population or a composition according to the invention in one or more pharmaceutically effective amounts or doses.
  • the desired dose may conveniently be presented in a single dose or as divided doses (which can again be subdosed) administered at appropriate intervals.
  • An administration regimen could include long-term (i.e., at least two weeks, and for example several months or years) or daily treatment.
  • an administration regimen can vary between once a day to once a month, such as between once a day and once every two weeks, such as but not limited to once a week.
  • a label comprising the same as disclosed herein may be administered once or several times, also intermittently, for instance on a daily basis for several days, weeks or months and in different dosages.
  • the amount applied of the label disclosed herein depends on the nature of the particular cancer disease. Multiple administrations are preferred.
  • radiolabelled materials are typically administered at intervals of 1 to 20 weeks apart or 2 to 10 weeks apart or 2 to 8 weeks apart or 3 to 6 weeks apart or 3 to 5 weeks apart or each 4 weeks.
  • the skilled artisan knows however how to choose dividing the administration into two or more applications, which may be applied shortly after each other, or at some other predetermined interval ranging e.g. from 1 day to 4 weeks.
  • the labels as envisaged herein may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained.
  • examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.
  • both treatments are applied to the patient in temporal proximity.
  • both treatments are applied to the patient within four weeks (28 days). More preferably, both treatments are applied within two weeks (14 days), more preferred within one week (7 days).
  • the two treatments are applied within two or three days.
  • the two treatments are applied at the same day, i.e. within 24 hours. In another embodiment, the two treatments are applied within four hours, or two hours, or within one hour.
  • the two treatments are applied in parallel, i.e. at the same time, or the two administrations are overlapping in time.
  • the populations or compositions according to the invention are applied together with a compound that is able to optimize and therefore reduce kidney retention of the label comprised in said populations or compositions.
  • “applied together with” is to be construed broadly. It means it encompasses applied simultaneously on one or in two distinct compositions. It also encompasses applied sequentially in two distinct compositions.
  • Such a compound may be a plasma or blood substitute such as modified gelatin.
  • modified gelatin such as GelofusineTM.
  • the use of such plasma or blood substitute is expected to optimize and therefore reduce the retention of the label in the kidney and therefore to optimize unwanted side effects.
  • Such a compound may be a positively charged amino acid or a composition comprising at least one positively charged amino acid.
  • suitable positively charged amino acids are arginine, lysine and/or histidine.
  • An example of such a composition is AminomedixTM The use of positively charged amino acids has been extensively described in WO 2014/204854 which is explicitly incorporated by reference.
  • the populations or compositions according to the invention are applied together with immunotherapy.
  • immunotherapy In an embodiment, with one or more therapeutic antibodies or therapeutic antibody fragments.
  • the label comprised in populations or compositions according to the invention and the one or more therapeutic antibodies or therapeutic antibody fragments may be infused at the same time, or the infusions may be overlapping in time. If they are administered at the same time, they may be formulated together in one single pharmaceutical preparation, or they may be mixed together immediately before administration from two different pharmaceutical preparations, for example by dissolving or diluting into one single infusion solution. In another embodiment, the two are administered separately, i.e. as two independent pharmaceutical compositions. In one preferred embodiment, administration of the two treatments is in a way that tumour cells within the body of the patient are exposed to effective amounts of the cytotoxic drug and the radiation at the same time. In another preferred embodiment, effective amounts of both compounds are present at the site of the tumour at the same time.
  • the present invention also embraces the use of further agents, which are administered in addition to the populations or compositions according to the invention as defined.
  • This could be, for example, one or more further chemotherapeutic agent(s). It could also be one or more agent(s) applied to prevent, suppress, or ameliorate unwanted side effects of any of the other drugs given.
  • a cytokine stimulating proliferation of leukocytes may be applied to ameliorate the effects of leukopenia or neutropenia.
  • the efficacy of the populations or compositions according to the invention can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person.
  • determining As used herein, the terms ‘determining’, ‘measuring’, ‘assessing’, ‘monitoring’ and ‘assaying’ are used interchangeably and include both quantitative and qualitative determinations.
  • the term 'prevention and/or treatment' comprises preventing and/or treating a certain disease and/or disorder and/or condition, preventing the onset of a certain disease and/or disorder and/or condition, slowing down or reversing the progress of a certain disease and/or disorder and/or condition, preventing or slowing down the onset of one or more symptoms associated with a certain disease and/or disorder and/or condition, reducing and/or alleviating one or more symptoms associated with a certain disease and/or disorder and/or condition, reducing the severity and/or the duration of a certain disease and/or disorder and/or condition, and generally any prophylactic or therapeutic effect of the populations or compositions according to the invention that is beneficial to the subject or patient being treated.
  • tumour metastatic cell
  • tumour cell refers to a cell that is present in a primary or metastatic tumour lesion.
  • tumours consist not only of cancer cells, but should be considered as organ-like structures in which a complex bidirectional interplay exists between transformed and non-transformed cells.
  • the malignant potential of transformed cells requires an apt support structure from the stroma, which can consist of fibroblasts, adipocytes, blood and lymph vessels, but may also be considerably infiltrated by a wide range of immune cells.
  • a tumour cell may also be a fibroblast, preferably a CAF.
  • solid tumour(s) or “tumour(s)” are meant primary tumours and/or metastases (wherever located).
  • solid cancer and solid tumor are used interchangeably herein. Examples of solid tumors are sarcomas, carcinomas, and lymphomas.
  • Examples of a sarcoma are an osteosarcoma or osteogenic sarcoma, a chondrosarcoma, a leiomyosarcoma, a rhabdomyosarcoma, a mesothelial sarcoma or mesothelioma, a fibrosarcoma, an angiosarcoma or hemangioendothelioma, a liposarcoma, a glioma or astrocytoma, a myxosarcoma, and a mesenchymous or mixed mesodermal tumor.
  • carcinomas are adenocarcinomas and squamous cell carcinomas.
  • Examples of lymphomas are non-Hodgkin lymphomas and Hodgkin lymphomas.
  • cancer cell refers to a cell that divides and reproduces abnormally and limitlessly with uncontrolled growth and which can break away and travel to other parts of the body and set up another site, referred to as metastasis.
  • a ’lesion’ as used herein can referto any abnormal change in a body tissue or organ resulting from injury or disease.
  • lesion typically refers to a tumour.
  • primary tumour(s) as used herein is a tumour growing at the anatomical site where tumour progression began and proceeded to yield a cancerous mass.
  • metastatic lesion(s) refers to malignant, or cancerous, tumours that have spread from their original location to other parts of the body.
  • Related medical terms that might be used interchangeably include late-stage cancer, advanced cancer, or metastatic disease.
  • metastatic lesions are considered to be incurable, although treatment is often available to control the spread of cancerous cells and potentially increase the individual's life expectancy.
  • Metastasis is the term for the spread of cancer beyond its originating site in the body.
  • metastatic lesions are cancerous tumours that are found in locations apart from the original starting point of the primary tumour. Metastatic tumours occur when cells from the primary tumour break off and travel to distant parts of the body via the lymph system and blood stream. Alternately, cells from the original tumour could seed into new tumours at adjacent organs or tissues.
  • Metalastatic disease refers to late-stage cancer and to the medical classification of cancer as being in stage III, when cancer cells are found in lymph nodes near the original tumour, or in stage IV, when cancer cells have travelled far beyond the primary tumour site to distant parts of the body. Metastatic lesions are most commonly found in the brain, lungs, liver, or bones.
  • An individual with metastatic cancer might or might not experience any symptoms, and the symptoms could be related to the area where metastasized cells have relocated. Once metastatic lesions are present in the body, the individual's cancer will be considered incurable for most cancer types. This means it is excessively difficult to eradicate every existing cancer cell with available treatments. In this case, the goal of treatment becomes slowing the growth of tumours to maintain the highest possible quality of life and potentially extend the individual's life expectancy. In some cases, people with metastatic lesions can live for a number of years with appropriate treatment for symptom management.
  • radionuclide As used herein, the terms ‘radionuclide’, ‘radioactive nuclide’, ‘radioisotope’ or ‘radioactive isotope’, are used interchangeably herein and refer to atoms with an unstable nucleus, characterized by excess energy available to be imparted either to a newly created radiation particle within the nucleus or via internal conversion. During this process, the radionuclide is said to undergo radioactive decay, resulting in the emission of gamma ray(s) and/or subatomic particles such as alpha or beta particles. These emissions constitute ionizing radiation. Radionuclides occur naturally or can be produced artificially.
  • immunohistochemistry refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in sections of biological tissues. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumours. IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
  • antigens e.g., proteins
  • Example 1 Methods according to the invention
  • VHH storage buffer (10 mM sodium acetate, 125 mM NaCI, pH 5.3 for anti-FAP VHH1, PBS for anti-HER2 VHH4) is brought to 18-25 °C
  • reaction mixture is formulated on an ultrafiltration device with a UF membrane area of 0.2 m 2 with 5kDa cut off.
  • the goal of this ELISA binding assay is to compare the EC50 of the different DOTA-VHH conjugates at different CARs, corresponding to the functional fraction that is able to bind the target antigen (FAP recombinant protein), using a reference batch as calibrator.
  • FAP recombinant protein target antigen
  • ELISA protocol A 96-well ELISA plate (Nunc Maxisorp flat-bottom plate, Invitrogen) was coated overnight at 4°C with 100 pl per well human FAP recombinant protein (Sino Biological) at 1 pg/ml in 100 mM NaHCOs, pH 8.2. The wells were overcoated with protein-free T20 (PBS) blocking buffer (Pierce). After washing with PBS, pH 7.4, 0.1% Tween, the VHH samples were added to every well.
  • PBS protein-free T20
  • VHH binding of VHHs was detected by using a rabbit anti-VHH polyclonal (custom made antiserum raised specifically against VHH1 ) as the primary Ab and goat anti-rabbit IgG-heavy and light chain antibody HRP conjugate (Bioke) as the secondary Ab, with thorough washing with PBS, pH 7.4, 0.1% Tween in between. Signals were developed using 3,3',5,5'-Tetramethylbenzidine (TMB) substrate (Sigma-Aldrich), and the development stopped after the addition of 1 M H2SO4.
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • n CAR
  • Results can be found in Figure 7. Overall, a low uptake can be seen in healthy organs, except for the kidneys. Relatively high uptake is seen in tumors for all compounds.
  • Human HER2 recombinant protein (Sino Biological) was diluted in 10 mM sodium acetate pH 4.5 and immobilized using the amine coupling kit (Cytiva) on a CM5 Biacore chip (Cytiva) with a target level of 740 RU.
  • the analytes were diluted to a concentration of 125 nM in HBS (10 mM Hepes, 150 mM NaCI, 3.4 mM EDTA, 0.005% Tween20, pH 7.4) and subsequently a 2-fold serial dilution series was prepared in HBS ranging from 125 nM until 0.488 nM.
  • a multi-cycle kinetics experiment was conducted with an analyte flow rate of 30 pl/min.
  • the association phase took 180 s and the dissociation phase 600 s.
  • Regeneration was done by two pulses of 100 mM glycine-HCI, pH 2.0 for 15 s at 30 pl/min, followed by 180 s stabilization.
  • Binding curves were fitted using a ‘1 :1 (antigen:analyte)’ binding model in Biacore T200 evaluation software.
  • Table 3 shows an overview of the different DOTA-VHH4 CAR species compared to an unconjugated reference. A slight increase in KD (M) can be observed with increasing CAR levels.
  • n CAR
  • Results can be found in Figure 3. It can be seen that there is no significant uptake of the compounds in healthy organs, except for the kidneys and urinary bladder. There is a significant difference in kidney uptake between the compounds. Higher CAR values correspond to less kidney uptake.
  • n CAR
  • the LAR of a population of labelled antibody fragments may be determined as follows.
  • the sample was thawed and 10-fold diluted in PBS to 0.3 pg/pl.
  • the mass spectrometer was operated in MS1 mode at a resolution of 60 000, a SID of 40 V, a spray voltage of 3.8kV, a capillary temperature of 320 °C, a sheat gas of 10, 3 microscans, an AGC target of 3E6, a maximum iontime of 200 ms and a mass range from 1000-3000 m/z in profile mode.
  • the spectra were deconvoluted using BioPharma Finder (Thermo Fisher Scientific) from minute 8.0 to 8.7, which corresponds to the elution time of the sdAb.
  • the output mass range was set from 8000 to 25000 Da, the signal to noise threshold on 3 and the minimum number of detected charges was set to 2.
  • the general ‘Protein’ isotope table was used. For more advanced deconvolution parameters we refer to the detailed PDF report generated by BioPharma Finder.
  • the deconvoluted spectra represent the monoisotopic mass of the detected ions.

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Abstract

La présente invention concerne un procédé de préparation d'une population de fragments d'anticorps marqués, ladite population comprenant des fragments d'anticorps et un certain nombre d'étiquettes, lesdits fragments d'anticorps comprenant un certain nombre de sites de conjugaison qui sont capables de se conjuguer auxdites étiquettes, chaque étiquette étant conjuguée à l'un desdits sites de conjugaison, le rapport dudit nombre d'étiquettes audit nombre desdits sites de conjugaison étant supérieur ou égal à 0,25, le procédé comprenant les étapes consistant à : a) fournir lesdits fragments d'anticorps ; b) fournir lesdites étiquettes ; c) faire réagir lesdites étiquettes avec lesdits fragments d'anticorps dans une solution tampon aqueuse, le pH de ladite solution tampon aqueuse étant choisi pour obtenir un rapport donné dudit nombre d'étiquettes audit nombre de sites de conjugaison, le pH de ladite solution tampon aqueuse étant de 9,0 jusqu'à 12,0.
PCT/EP2023/060283 2022-04-22 2023-04-20 Anticorps radiomarqué amélioré WO2023203135A1 (fr)

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