Ph-dependent Anti-cd22 antibody or antibody fragment

Abstract

The present invention relates to engineered proteins that can be used as platforms to deliver cytotoxic drugs, imaging labels or other cargo molecules to target cells such as tumour cells. These proteins are engineered to deliver the cargo molecules more efficiently to late endosomes and lysosomes in the targeted cells.

Description

AN ANTIBODY OR ANTIBODY FRAGMENTField of the Invention

[0001] The present invention relates to engineered proteins that can be used as platforms to deliver cytotoxic drugs, imaging labels or other cargo molecules to target cells such as tumour cells. These proteins are engineered to deliver the cargo molecules more efficiently to late endosomes and lysosomes in the targeted cells.

[0002] Antibody-drug conjugates (ADCs) or protein-drug conjugates (PDCs) represent a class of therapeutics that combine the high specificity of antibodies, antibody fragments or other proteins that bind to cancer cells or other unwanted cells such as inflammatory or virally infected cells with the delivery of highly toxic drugs. A problem with current ADCs or PDCs is that they have toxicities that can limit the dose. However, the development of ADCs and PDCs that allow more efficient delivery of cytotoxic drugs to tumour cells remains challenging.

[0003] In addition, antibodies, antibody fragments or other targeting proteins can be labelled with radiolabel, fluorescent label or near infrared label to be used as labelled conjugates (LCs) in diagnostic imaging. However, the development of LCs that allow more efficient labelling of tumours with higher contrast to background tissues also remains challenging.

[0004] CD22 is a marker on B cell malignancies. To date, an ADC (Besponsa, or inotuzumab ozogamicin) that targets CD22 has been approved (2017) to treat B cell acute lymphoblastic leukaemia. However, this ADC is associated with significant off-target toxicities that lead to hepatotoxicity. Other adverse side effects reported in greater than 20% patients include thrombocytopenia, neutropenia, infection, anemia, leukopenia, fatigue, hemorrhage, pyrexia, nausea and headache.

[0005] To be effective in killing cells, ADCs need to deliver their cytotoxic payload to the degradative compartments, namely lysosomes, in cells.Brief Summary of the Disclosure

[0006] The invention involves the generation of an antibody-drug conjugate (ADC) that has improved drug delivery properties. The inventors have taken the approach of engineering the antibody component of the ADC so that it delivers its cytotoxic payload more efficiently to lysosomes. This is achieved by engineering the antibody (in this case, inotuzumab) so that itbinds to CD22 with substantially higher affinity at near neutral pH (pH 7.4) than at the acidic pH (around pH 6.0) in endosomes within cells. This binding behaviour leads to uptake into CD22-expressing cells, following by dissociation from CD22 within endosomes and delivery into lysosomes. The inventors have shown that such ‘acid-switched’ ADCs are more effective in delivering ADC / cytotoxic drugs to cancer cells, indicating that they can be used at much lower doses to achieve anti-tumour effects. In turn, this is expected to result in reduced toxicities towards normal tissues / organs and the ability to target tumours with a wide range of CD22 expression levels. The engineered ADC that the inventors have generated to target CD22 has lower off-target toxicities. The toxicities associated with the approved ADCs are dose-limiting, leading to reduced therapeutic effects and inefficient targeting of tumours with low-intermediate expression levels of target. This not only leads to undesirable side effects, but can also result in escape of tumour cells expressing reduced target levels.

[0007] The ADC may be used to treat B cell malignancies. The incidence of non-Hodgkin lymphoma (for which diffuse B cell lymphoma and follicular lymphoma constitute about 30% and 20% of cases, respectively) is about 70,000 cases per year in each of the US and Western Europe (Kanas et al., Leukemia & Lymphoma, 63, 54-63, 2022). Numerous cells lines exhibit characteristics of B lymphocytes and can be representative of B-cell cancers in general. Positive responses in such cell lines are indicative of positive therapeutic responses for more general B-cell cancers, such as the currently claimed B-cell cancers, such as B-cell malignancies, B cell acute lymphoblastic leukaemia, non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B cell lymphoma These cell lines include: Daudi cells (B lymphoblasts isolated from the peripheral blood of a Burkitt's Lymphoma patient), Raji cells (lymphoblast-like cells established from a Burkitt's lymphoma), Bjab cells (a B cell line derived from Burkitt's lymphoma), Ramos cells (a B lymphocyte cell line derived from a Burkitt's lymphoma), Reh cells (a human cell line exhibiting lymphoblastic morphology that was isolated from tissue from an acute lymphocytic leukemia (ALL) patient), SU-DHL-4 cells (a cell line from a patient with diffuse large B-cell lymphoma), and DOHH-2 cells (a cell line from a B cell lymphoma). Furthermore, CD22 expressing cells and CD22 expressing tumours are suitable models for B-cell cancers in general, as CD22 is a B-cell marker expressed in most B-cell malignancies, including B-cell acute lymphoblastic leukemia (B-ALL), mantle cell lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma.

[0008] The invention was developed through the concept that the engineering of antibodies to have high affinity binding to target at near neutral pH, and very low affinity at endosomal acidic pH, would serve as very efficient lysosomal delivery vehicles for ADCs. Proof of concept for this has been achieved by engineering the CD22-specific antibody, inotuzumab,for such acid-switched binding behaviour and using the engineered antibody to generate an ADC. Compared with an ADC comprising the parent antibody (wild type inotuzumab), the resulting ADC accumulates to substantially higher levels in lysosomes in target cells and is more cytotoxic towards tumour cells. The inventors have named ADCs of this class ALTAs, for ADCs with increased Lysosomal Trafficking Activity.

[0009] In the antibodies and antibody fragments of the present invention, histidine mutations are used to make pH-dependent antibodies. The inventors surprisingly developed antibodies (discussed as Mutant A in the examples) that not only have mutations to histidine in VHCDR3 and VLCDR3, but that also have mutations of Tyr-Thr to Val-Val in VLCDR3. Given the lack of charge and hydrophobicity of these residues, the skilled person would not have expected this combination of mutated residues to lead to antibodies with increased pH dependence for binding.

[0010] In a first aspect, the invention provides an endolysosomal targeting conjugate, comprising: a) a targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to cell surface CD22 of a target cell with a lower dissociation constant in an extracellular space than in an endolysosomal compartment of the target cell; and b) a cargo component comprising a cargo molecule; wherein the targeting component is fused directly or indirectly to the cargo component; wherein upon entry to the endolysosomal compartment, the targeting component is configured to dissociate from the cell surface CD22; and wherein the endolysosomal targeting conjugate is configured to deliver the cargo molecule to the endolysosomal compartment of the target cell.

[0011] In other words, the invention provides an endolysosomal targeting conjugate, comprising: a) a targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to cell surface CD22 of a target cell with a lower dissociation constant in an extracellular space than in an endolysosomal compartment of the target cell; and b) a cargo component comprising a cargo molecule conjugated to the targeting component (i.e. the targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain); wherein the targeting component is fused directly or indirectly to the cargo component; wherein upon entry to the endolysosomal compartment, the targeting component is configured to dissociate from the cell surface CD22; and wherein the endolysosomal targeting conjugate is configured to deliver the cargo molecule to the endolysosomal compartment of the target cell.

[0012] In a second aspect, the invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0013] In another aspect, the invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0014] In a further aspect, the invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 7; and b) a light chain variable region (VL) comprising a lightchain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 8.

[0015] In a third aspect, the invention provides a nucleic acid encoding the antibody or antibody fragment of the invention.

[0016] In a further aspect, the invention provides a nucleic acid encoding the antibody or antibody fragment of the invention, or the targeting component of the endolysosomal targeting conjugate of the invention.

[0017] In a fourth aspect, the invention provides an expression vector comprising the nucleic acid of the invention.

[0018] In a fifth aspect, the invention provides a host cell comprising the expression vector of the invention.

[0019] In a sixth aspect, the invention provides a pharmaceutical composition comprising the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, and / or the host cell of the invention; and further comprising a pharmaceutically acceptable carrier.

[0020] In another aspect, the invention provides a pharmaceutical composition comprising the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, and / or the host cell of the invention; and further comprising a pharmaceutically acceptable carrier.

[0021] In a seventh aspect, the invention provides the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in therapy.

[0022] In another aspect, the invention provides the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in therapy.

[0023] In an eighth aspect, the invention provides the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in treating cancer.

[0024] In another aspect, the invention provides the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in treating cancer.

[0025] In a ninth aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention, in a therapeutically effective amount, to treat the cancer.

[0026] In another aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention, in a therapeutically effective amount, to treat the cancer.

[0027] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0028] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0029] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0030] Various aspects of the invention are described in further detail below.Brief Description of the Figures

[0031] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings.

[0032] Table 1 - Determination of equilibrium dissociation constants (KDS) using surface plasmon resonance (SPR; BIAcore) of the interactions of wild type (WT) antibody and mutants A, B and C as Fab fragments with immobilised extracellular domain of humanCD22. Fab fragments were diluted in PBS-T buffer (PBS containing 0.01% Tween-20) with pH adjusted to either pH 7.4 or pH 6.0 at concentrations ranging from 5-2560 nM (0.005- 2.56 mM) and injected at a flow rate of 5 pl / minute. The equilibrium dissociation constants (KDS) at pH 7.4 or pH 6.0 were determined. The CD22-coupled flow cell of the chip was regenerated between runs by injecting 50 mM borate, pH 11, 0.5 M sodium chloride for 60 seconds at a flow rate of 30 pl / minute.

[0033] Figure 1 - Accumulation of WT-MMAE and Mut A-MMAE in CD22-expressing tumour cell lines. Cells were treated with 30 pg / ml Alexa 488-labelled WT-MMAE or Mut A- MMAE (DAR=2) in growth medium for 0.5, 4 and 18 hours at 37°C in a 5% CO2 incubator. The cells were cooled on ice and incubated with 25 pg / ml Alexa Fluor 488-specific antibody for 30 minutes to quench the Alexa Fluor 488 signal associated with surface-bound ADC. The cells were then washed and analysed using a Cytoflex-S flow cytometer (Beckman Coulter). The quenching efficiency was determined using separate cell samples which were incubated with Alexa 488-labelled antibodies on ice for 30 minutes followed by treatment with Alexa Fluor 488-specific quenching antibody. Mean values for triplicate samples are shown. Error bars indicate SD, and statistically significant differences are shown (two-way ANOVA, Bonferroni correction for multiple comparisons; *p < 0.05, **p < 0.01 , ***p < 0.001).

[0034] Figure 2 - Internalisation of WT-MMAE and Mut A-MMAE (DAR = 2) and analysis of lysosomal delivery in CD22-expressing tumour cell lines. Cells were pulsed and chased with Alexa Fluor 647-labelled dextran (0.25 mg / ml) for 2 and 3 hours respectively (37°C, 5% CO2), followed by treatment with 0.03 mg / ml Alexa Fluor 555-labelled WT-MMAE or Mut A- MMAE for 4 and 18 hours as indicated (37°C, 5% CO2). Cells were then washed and fixed. Fluorescence images were acquired using a Zeiss Axio Observer 7 fluorescence microscope (Objective: 100x 1.4NA Zeiss. Camera: Hamamatsu ORCA Flash 4.0 V3. Light source: Xylis-X-Cite LED illuminator XT720S. Filter Sets: Semrock Brightline; Alexa Fluor 555: FF562; Alexa Fluor 647: FF660). Astero Lumio software (Astero Technologies LLC) was used with piecewise linear adjustment for image analysis. Scale bars represent 1 pm.

[0035] Figure 3 - Cytotoxicity of ADCs comprising WT antibody and mutant A (Mut A) towards CD22-expressing tumour cells. Cells were treated with ADCs conjugated with MMAE with a DAR of two (2C) (A), four (4C) (B) or Deruxtecan (DXd) with a DAR of two (2C) (C) or four (4C) (D). As a control, a MMAE-conjugated ADC (DAR = 2) comprising a hen egg lysozyme-specific antibody (HuLyslO) was used. Following incubation for 72 hours, cell viability was determined using an MTS assay. Data shown represent means of optical density (OD; 490 nm). Two-way ANOVA with Bonferroni correction for multiple comparison was used for statistical analyses, and statistical significance is shown for Mut A vs. corresponding WT ADCs (*p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001).

[0036] Figure 4 - Female SCID mice bearing CD22-expressing tumours were treated weekly (three times; arrowheads) with 4 mg / kg ADC [WT-MMAE (2C) or Mut A-MMAE (2C)] or vehicle (PBS) (n= 5-7 mice per group). The mean tumour volume for each treatment group is shown and error bars indicate SEM (A). Statistically significant differences at the experimental endpoints are indicated (two-tailed Mann-Whitney ll-test and unpaired two- tailed t-test; **p < 0.01 , ***p < 0.001). The tumour volume for each treatment group with individual mice is shown (B).

[0037] Figure 5 - Accumulation of WT-MMAE, Mut A-MMAE, Mut B-MMAE or Mut C- MMAE in CD22-expressing tumour cell lines. Cells were treated with 30 pg / ml Alexa 488- labelled WT-MMAE, Mut A-MMAE, Mut B-MMAE or Mut C-MMAE (DAR=2) in growth medium for 4 and 18 hours at 37oC in a 5% CO2 incubator. The cells were cooled on ice and incubated with 25 pg / ml Alexa Fluor 488-specific antibody for 30 minutes to quench the Alexa Fluor 488 signal associated with surface-bound ADC. The cells were then washed and analysed using a Cytoflex-S flow cytometer (Beckman Coulter). The quenching efficiency was determined using separate cell samples which were incubated with Alexa 488-labelled antibodies on ice for 30 minutes followed by treatment with Alexa Fluor 488-specific quenching antibody. Mean values for triplicate samples are shown. Error bars indicate SD, and statistically significant differences are shown (two-way ANOVA, Bonferroni correction for multiple comparisons; **p < 0.01, ***p < 0.001).

[0038] Figure 6 - Accumulation of WT-MMAE or Mut A-MMAE (A) and WT-DXd or Mut A- DXd (B) in CD22-expressing tumour cell lines. Cells were treated with 30 pg / ml Alexa 488- labelled WT-MMAE, Mut A-MMAE WT-DXd or Mut A-DXd (DAR=2) in growth medium for 4 and 18 hours at 37°C in a 5% CO2 incubator. The cells were cooled on ice and incubated with 25 pg / ml Alexa Fluor 488-specific antibody for 30 minutes to quench the Alexa Fluor 488 signal associated with surface-bound ADC. The cells were then washed and analysed using a Cytoflex-S flow cytometer (Beckman Coulter). The quenching efficiency was determined using separate cell samples which were incubated with Alexa 488-labelled antibodies on ice for 30 minutes followed by treatment with Alexa Fluor 488-specific quenching antibody. Mean values for triplicate samples are shown. Error bars indicate SD, and statistically significant differences are shown (two-way ANOVA, Bonferroni correction for multiple comparisons; **p < 0.01, ***p < 0.001).

[0039] Figure 7 - Internalisation of WT-MMAE and Mut A-MMAE (DAR = 2) and analysis of lysosomal delivery in CD22-expressing tumour cell lines. Cells were pulsed and chased with Alexa Fluor 647-labelled dextran (0.25 mg / ml) for 2 and 3 hours respectively (37°C, 5% CO2), followed by treatment with 30 pg / ml Alexa Fluor 555-labelled WT-MMAE or Mut A- MMAE for 4 and 18 hours as indicated (37°C, 5% CO2). Cells were then washed and fixed.Fluorescence images were acquired using a Zeiss Axio Observer 7 fluorescence microscope (Objective: 100x 1.4NA Zeiss. Camera: Hamamatsu ORCA Flash 4.0 V3. Light source: Xylis-X-Cite LED illuminator XT720S. Filter Sets: Semrock Brightline; Alexa Fluor 555: FF562; Alexa Fluor 647: FF660). Astero Lumio software (Astero Technologies LLC) was used with piecewise linear adjustment for image analysis. Scale bars represent 1 pm.

[0040] Figure 8 - Cytotoxicity of ADCs comprising WT antibody and mutant A (Mut A) towards CD22-expressing tumour cells. Cells were treated with ADCs conjugated with MMAE with a DAR of two (2C) (A) or conjugated with Deruxtecan (DXd) with a DAR of two (2C) (B). Following incubation for 72 hours, cell viability was determined using an MTS assay. Data shown represent means of optical density (OD; 490 nm). Two-way ANOVA with Bonferroni correction for multiple comparison was used for statistical analyses, and statistical significance is shown for Mut A vs. corresponding WT ADCs (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0041] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0042] Various aspects of the invention are described in further detail below.Detailed Description

[0043] In a first aspect, the invention provides an endolysosomal targeting conjugate, comprising: a) a targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to cell surface CD22 of a target cell with a lower dissociation constant in an extracellular space than in an endolysosomal compartment of the target cell; and b) a cargo component comprising a cargo molecule; wherein the targeting component is fused directly or indirectly to the cargo component; wherein upon entry to the endolysosomal compartment, the targeting component is configured to dissociate from the cell surface CD22; and wherein the endolysosomal targeting conjugate is configured to deliver the cargo molecule to the endolysosomal compartment of the target cell.

[0044] In other words, the invention provides an endolysosomal targeting conjugate, comprising: a) a targeting component comprising an antibody, an antibody fragment, anantibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to cell surface CD22 of a target cell with a lower dissociation constant in an extracellular space than in an endolysosomal compartment of the target cell; and b) a cargo component comprising a cargo molecule conjugated to the targeting component (i.e. the targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain); wherein the targeting component is fused directly or indirectly to the cargo component; wherein upon entry to the endolysosomal compartment, the targeting component is configured to dissociate from the cell surface CD22; and wherein the endolysosomal targeting conjugate is configured to deliver the cargo molecule to the endolysosomal compartment of the target cell.

[0045] In a second aspect, the invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0046] In another aspect, the invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising the following mutations: i) Asparagine(N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0047] In a further aspect, the invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 7; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 8.

[0048] In a third aspect, the invention provides a nucleic acid encoding the antibody or antibody fragment of the invention.

[0049] In a further aspect, the invention provides a nucleic acid encoding the antibody or antibody fragment of the invention, or the targeting component of the endolysosomal targeting conjugate of the invention.

[0050] In a fourth aspect, the invention provides an expression vector comprising the nucleic acid of the invention.

[0051] In a fifth aspect, the invention provides a host cell comprising the expression vector of the invention.

[0052] In a sixth aspect, the invention provides a pharmaceutical composition comprising the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, and / or the host cell of the invention; and further comprising a pharmaceutically acceptable carrier.

[0053] In another aspect, the invention provides a pharmaceutical composition comprising the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, and / or the host cell of the invention; and further comprising a pharmaceutically acceptable carrier.

[0054] In a seventh aspect, the invention provides the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the hostcell of the invention and / or the pharmaceutical composition of the invention for use in therapy.

[0055] In another aspect, the invention provides the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in therapy.

[0056] In an eighth aspect, the invention provides the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in treating cancer.

[0057] In another aspect, the invention provides the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in treating cancer.

[0058] In a ninth aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention, in a therapeutically effective amount, to treat the cancer.

[0059] In another aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the subject the antibody or antibody fragment of the invention, the endolysosomal targeting conjugate of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention, in a therapeutically effective amount, to treat the cancer.

[0060] Suitably, the endolysosomal targeting conjugate of the invention may comprise wherein the targeting component is configured to bind to cell surface CD22 in the extracellular space with a dissociation constant less than 500 nM.

[0061] Suitably, the endolysosomal targeting conjugate of the invention may comprise wherein the targeting component is configured to bind to cell surface CD22 with a lower dissociation constant at a near neutral pH than at an acidic endolysosomal pH.

[0062] Suitably, the endolysosomal targeting conjugate of the invention may comprise wherein the near neutral pH is from about 6.8 to about 7.5 and the acidic endolysosomal pH is from about 5.0 to about 6.5.

[0063] Suitably, the endolysosomal targeting conjugate of the invention may comprise wherein the targeting component comprises a Fab fragment or a scFv fragment of a CD22- specific antibody, wherein the Fab fragment or the scFv fragment comprises: a) a heavy chain variable domain of SEQ ID NO. 9 further comprising a mutation of Asn103 to histidine and / or b) a light chain variable domain of SEQ ID NO. 10 further comprising a mutation of Gln99 to histidine, Tyr101 to valine and / or Thr102 to valine.

[0064] Suitably, the antibody or antibody fragment of the invention may comprise: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0065] Suitably, the antibody or antibody fragment of the invention may comprise: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0066]

[0067] Suitably, the antibody or antibody fragment of the invention may comprise: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11 ; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 12.

[0068] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment further has mutated amino acid residues as follows: a) position Cys220 mutated to Ser220 and Cys229 mutated to Ser229 in the heavy chain; and Cys214 mutated to Ser214 in the light chain; or b) Cys220 mutated to Ser220 in the heavy chain and Cys214 mutated to Ser214 in the light chain; according to EU numbering.

[0069] Suitably, the antibody or antibody fragment of the invention may comprise drug to antibody ratios (DARs) of a) two; or b) four.

[0070] Suitably, the antibody or antibody fragment of the invention may comprise a heavy chain hinge region comprising the amino acid sequence of a) SEQ ID NO: 14; or b) SEQ ID NO: 17.

[0071] Suitably, the antibody or antibody fragment of the invention may comprise a heavy chain comprising the amino acid sequence of a) SEQ ID NO: 13; or b) SEQ ID NO: 16.

[0072] Suitably, the antibody or antibody fragment of the invention may comprise a light chain comprising the amino acid sequence of SEQ ID NO: 19.

[0073] Suitably, the antibody fragment of the invention may comprise wherein the fragment is selected from the group consisting of antigen-binding fragments (Fab), F(ab’)2, single chain variable fragments (scFv), an antibody domain, a nanobody and “third generation” (3G) fragments.

[0074] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment is a human antibody or antibody fragment.

[0075] Suitably, the antibody of the invention may comprise wherein the antibody is a full length antibody.

[0076] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment comprises a heavy chain constant region selected from lgG1, lgG2, lgG3 and lgG4, preferably lgG1 and lgG4, most preferably lgG1.

[0077] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment comprises FcgR-ablating mutations.

[0078] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment comprises a light chain constant region of kappa.

[0079] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment is monospecific.

[0080] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment is bispecific.

[0081] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment is humanised, optimised, de-immunized and / or conjugated.

[0082] Suitably, the antibody or antibody fragment of the invention may comprise wherein the antibody or antibody fragment is conjugated to a cargo molecule.

[0083] Suitably, the antibody or antibody fragment of the invention may comprise wherein the cargo molecule is an imaging label.

[0084] Suitably, the antibody or antibody fragment of the invention may comprise wherein the imaging label is a radiolabel or a fluorescent or a near infrared label.

[0085] Suitably, the antibody or antibody fragment of the invention may comprise wherein the cargo molecule is a drug and / or a linker.

[0086] Suitably, the antibody or antibody fragment of the invention may comprise wherein the drug is a cytotoxic drug.

[0087] Suitably, the antibody or antibody fragment of the invention may comprise wherein the cytotoxic drug is maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl- monomethyl auristatin E (MMAE).

[0088] Suitably, the antibody or antibody fragment of the invention may comprise wherein the cytotoxic drug is deruxtecan.

[0089] Suitably, the antibody or antibody fragment of the invention and / or the pharmaceutical composition of the invention may be for use according to the invention, wherein the antibody or antibody fragment of the invention is conjugated to a drug and / or a linker.

[0090] Suitably, the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention may be for use according to the invention, wherein the cancer is a tumour expressing CD22.

[0091] Suitably, the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention may be for use according to the invention, wherein the cancer is a B cell tumour expressing CD22.

[0092] Suitably, the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention may be for use according to the invention, wherein the cancer is selected from the group consisting of: B-cell malignancies, B cell acute lymphoblastic leukaemia, non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B cell lymphoma.

[0093] Suitably, the method of the invention may comprise wherein the antibody or antibody fragment of the invention is conjugated to a drug and / or a linker.

[0094] Suitably, the method of the invention may comprise wherein the cancer is selected from the group consisting of: B-cell malignancies, B cell acute lymphoblastic leukaemia, nonHodgkin lymphoma, follicular lymphoma, and diffuse large B cell lymphoma.

[0095] This disclosure relates to engineered antibodies or fusion proteins that are configured to allow improved delivery of a cargo molecule, such as a cytotoxic drug or an imaging label to late endosomes and lysosomes in a target cell. The terms "endolysosomal" and "endolysosomal compartment" as used herein refer to the early endosomes, late endosomes, lysosomes and associated tubulovesicular transport carriers of cells. Accordingly, the term "endolysosomal targeting conjugate" as used herein refers to an engineered antibody or fusion protein that is configured for improved delivery of a cargo molecule to the endolysosomal compartment of a target cell.

[0096] The antibodies or fusion proteins described herein include a targeting component and a cargo component.

[0097] The endolysosomal targeting conjugate of the invention may comprise an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0098] The endolysosomal targeting conjugate of the invention may comprise an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1 , a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chainvariable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0099] The endolysosomal targeting conjugate of the invention may comprise an antibody or antibody fragment that binds to human CD22 comprising: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0100] The endolysosomal targeting conjugate of the invention may comprise an antibody or antibody fragment that binds to human CD22 comprising: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0101] The “targeting component” may include an antibody, an antibody fragment, antibody domain, nanobody, protein, protein fragment, or protein domain that is configured to bind to a cell surface molecule, such as a cell surface receptor or other cell surface molecule that may be present on the extracellular surface of a cell's plasma membrane, wherein the molecule is at least partially exposed to an extracellular space. In the present invention, the cell surface molecule is CD22 (i.e. cell surface CD22).

[0102] The targeting component of the endolysosomal targeting conjugate of the invention may comprise an antibody or antibody fragment that binds to human CD22 comprising: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0103] The targeting component of the endolysosomal targeting conjugate of the invention may comprise an antibody or antibody fragment that binds to human CD22 comprising: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0104] The targeting component described herein may comprise a Fab fragment or a scFv fragment of a CD22-specific antibody, comprising a heavy chain variable domain of SEQ ID NO. 9 further comprising a mutation of Asn103 to histidine. Further, the targeting component described herein may comprise wherein the targeting component comprises a Fab fragment or a scFv fragment of a CD22-specific antibody, comprising a heavy chain variable domain of SEQ ID NO. 9 further comprising a mutation of Asn103 to histidine and / or a light chain variable domain of SEQ ID NO. 10 further comprising a mutation of Gln99 to histidine, Tyr101 to valine and / or Thr102 to valine

[0105] The term “configured to bind” means that the targeting component has been designed such that it stably associates with the cell surface molecule.

[0106] The term “extracellular surface” means the surface of a cell situated or occurring on the outer side of the cell or outside the cell’s body. The outer surface of a cell is in the extracellular space. The term “extracellular space” or “extracellular environment” refers to everything outside a cell, but still within the organism. This space is usually taken to be outside the plasma membranes, and occupied by fluid.

[0107] The terms "antibody-drug conjugate" or "ADC" as used herein refer to antibodybased conjugates that are configured to deliver a drug to a cell. In particular, the terms "antibody-drug conjugate" or "ADC" as used herein refers to an antibody that includes a targeting component and a cargo component. The targeting component may include an antibody Fab fragment, an antibody variable domain, or a nanobody. The targeting component is linked to a cargo component that may comprise one or more cytotoxic drug molecules.

[0108] The terms "protein-drug conjugate", or "PDC" as used herein refer to protein-based conjugates that are configured to deliver a drug to a cell. In particular, the term "protein-drug conjugate" or "PDC" as used herein refers to a protein that includes a targeting component and a cargo component. The targeting component may include a protein that binds to a cell surface receptor or cell surface molecule, or a Fab fragment, an antibody variable domain, or a nanobody. The targeting component is linked to a cargo component that may comprise one or more cytotoxic drug molecules

[0109] The term "labeled conjugate" or "LC" as used herein refers to an antibody or protein that includes a targeting component linked to a cargo component. Accordingly, the targeting component may include an antibody Fab fragment, an antibody variable domain, nanobody, or a protein that binds to a cell surface receptor or cell surface molecule. The cargo component may comprise one or more imaging labels, such as radiolabels, fluorescent molecules or other labeled molecules.

[0110] In general, for ADCs or PDCs to effectively deliver a drug to a cell, they should selectively bind to target cells, be internalized into the cells, and enter degradative compartments called late endosomes and lysosomes. Prior to entering lysosomes, ADCs or PDCs enter early endosomes where sorting into the recycling or lysosomal pathway occurs. Improving delivery to late endosomes and lysosomes (the endolysosomal pathway) would result in more potent ADCs or PDCs and enable the use of lower dosages.

[0111] In addition, LCs that allow improved delivery of imaging labels to late endosomes and lysosomes in target cells would result in higher contrast for imaging target cells against a background of non-targeted cells or tissue.

[0112] Accordingly, the current invention generally relates to endolysosomal targeting conjugates that are engineered to more efficiently deliver cargo molecules to the endolysosomal compartment of target cells, whereby the engineered conjugates described herein are configured to respond to differences in chemical composition in endosomes or late endosomes and the extracellular environment, thereby achieving more efficient delivery of a cargo molecule such as a cytotoxic drug or an imaging label to target cells. Examples ofthe endolysosomal targeting conjugates described herein include improved ADCs, PDCs, and LCs that are configured for improved targeting of the conjugated cargo molecules such as drugs or imaging labels to the endolysosomal compartment of target cells, thereby resulting in more potent ADCs or PDCs, or higher contrast LCs.

[0113] Cargo molecules as described herein can include any molecule having a function useful to cause an effect in a target cell. For example, in addition to cytotoxic drugs or imaging labels, additional cargo molecules could be radiolabels that kill cells through radiation damage i.e. can be used therapeutically rather than for imaging. Examples of such radiolabels are Yttrium (Y)-90 and Iodine (I)- 131. Additional types of cargo molecules are identifiable by skilled persons upon reading the present disclosure.

[0114] In addition to tumour cells, “target cells” in the sense of the disclosure may include other types of unwanted cells, such as inflammatory cells, or virally-infected cells, among others identifiable by skilled persons upon reading the present disclosure.

[0115] The term "cell surface” molecule as used herein refers to a protein or other biological molecule (e.g. phospholipid, carbohydrate) that is at least partially exposed on the extracellular surface of a plasma membrane of a cell.

[0116] In the present invention, the cell surface molecule is cell surface CD22.

[0117] Target cells of the present invention comprise cells expressing cell surface CD22. CD22 is expressed on the surface of B-cell lineage cells from the early progenitor stage of pro-B cell until terminal differentiation to mature B cells. CD22 may also be expressed on tumour cells such as tumour cells in B-cell malignancies. The term “CD22-positive cell” refers to a cell that expresses CD22 on its surface. The term “CD22-positive cancer” refers to a cancer comprising cells that express CD22 on their surface.

[0118] A tumour is an abnormal growth of cells that may form a mass or lump, but may be distributed diffusely. A “tumour” can be a benign tumour or a malignant tumour. Benign tumours are those that stay in their primary location without invading other sites of the body. They do not spread to local structures or to distant parts of the body. Benign tumours are caused by the presence of noncancerous cells. Specific types of benign tumours can turn into malignant tumours. Malignant tumours are caused by the presence of cancerous cells or precancerous cells, i.e. cells that grow uncontrollably and can spread locally and / or to distant sites within the body. Malignant tumours have the ability to invade other sites of the body. They may spread to distant sites via the bloodstream or the lymphatic system. This spread is called metastasis.

[0119] The malignant tumour or neoplasm may comprise a mixture of cancerous cells (and / or pre-cancerous cells) and healthy (i.e. non-cancerous) cells. When a subject has a malignant tumour it can be said that the subject has cancer.

[0120] The term “tumour” as used herein, encompasses the tumour in its entirety, i.e. the cells present within the tumour including cancerous, pre-cancerous cells, and / or healthy cells (for example stromal cells), as well as the tumour microenvironment which typically comprises immune cells and interstitial fluid.

[0121] “Cancer cells” may be defined by one or more of the following characteristics: reduced differentiation, self-sufficiency in growth signalling, insensitivity to anti-growth signals, evasion of apoptosis, enabling of a limitless replicative potential, induction and sustainment of angiogenesis, and / or activation of metastasis and invasion of tissue.

[0122] As used herein, “pre-cancer” or a “pre-cancerous condition” is an abnormality that has the potential to become cancer (such a cancer mentioned hereinabove), wherein the potential to become cancer is greater than the potential if the abnormality was not present, i.e., was normal. Examples of pre-cancer include but are not limited to adenomas, hyperplasias, metaplasias, dysplasias, benign neoplasias (benign tumours), premalignant carcinoma in situ, and polyps. In one example, the pre-cancer is a pre-cancer tumour. Such a tumour may comprise pre-cancerous and healthy cells.

[0123] A cancer may be a solid cancer or a liquid cancer. Suitably, a cancer or the tumour may be selected from the group consisting of B-cell malignancies, B cell acute lymphoblastic leukaemia, non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B cell lymphoma. Suitably, a B-cell malignancy may be Burkitt's lymphoma.

[0124] An endolysosomal targeting conjugate may dissociate in early or late endosomes from the receptor or cell surface molecule due to lower (acidic) pH, lower Ca2+concentrations, lower Cl’ or Na+concentrations, higher K+concentrations or other environmental conditions that distinguish endosomes from the extracellular space (for example, as described in Scott, C.C., Gruenberg, J. (2010) Ion flux and the function of endosomes and lysosomes: pH is just the start. Bioessays 33, 103-110). Accordingly, in some examples of the endolysosomal targeting conjugates herein described, the affinity of the targeting component for the cell surface molecule is higher in the extracellular space than in the endolysosomal compartment. Thus, the targeting component and the cell surface molecule may bind with a lower dissociation constant in the extracellular space. The endolysosomal targeting conjugate is delivered to late endosomes or lysosomes, resulting in release of its drug or label.

[0125] The term “dissociation constant” or “KD” refers to a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, such as when a complex falls apart into its component molecules. Equilibrium dissociation constants (KDS) can be determined using surface plasmon resonance. The lower the dissociation constant, the more tightly bound the component molecules, or the higher the affinity between the component molecules.

[0126] According to some embodiments, Kd is measured using surface plasmon resonance assays using a BIACCRE®-2000 or a BIACCRE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at -500-1,000 response units (Rll).

[0127] In some embodiments, the endolysosomal targeting conjugate of the invention may comprise wherein the targeting component is configured to bind to cell surface CD22 with a lower dissociation constant at a near neutral pH than at an acidic endolysosomal pH. As described herein, a near neutral pH may be from about 6.8 to about 7.5 and an acidic endolysosomal pH may be from about 5.0 to about 6.5.

[0128] The targeting component and the cargo component may be linked by a covalent bond or may be non-covalently associated with each other.

[0129] In some embodiments, the endolysosomal targeting conjugate of the invention may comprise wherein the targeting component is configured to bind to cell surface CD22 in the extracellular space with a dissociation constant less than 500 nM.

[0130] Endolysosomal targeting conjugates described herein comprise targeting components that are configured to bind to human CD22. In particular, the exemplary endolysosomal targeting conjugates may bind to CD22 with affinities of less than 500 nM at near-neutral pH.

[0131] CD22 is a cell surface receptor that is overexpressed on tumours and is therefore a well-characterized tumour targets. It is found on the surface of mature B cells and to a lesser extent on some immature B cells. Generally speaking, CD22 is a regulatory molecule that prevents the overactivation of the immune system and the development of autoimmune diseases. CD22's main function is to inhibit B-cell receptor (BCR) signalling. CD22 is an endocytic receptor that recycles between the cell surface and the endosomes.

[0132] CD22 is a sugar binding transmembrane protein, which specifically binds sialic acid with an immunoglobulin (Ig) domain located at its N-terminus. The presence of Ig domains makes CD22 a member of the immunoglobulin superfamily. CD22 functions as an inhibitory receptor for B cell receptor (BCR) signaling.

[0133] The term “CD22,” as used herein, refers to any native CD22 from any vertebrate source, including mammals such as primates (e.g. humans, cynomolgus monkey (cyno)) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full- length,” unprocessed CD22 as well as any form of CD22 that results from processing in the cell. The term also encompasses naturally occurring variants of CD22, e.g., splice variants, allelic variants, and isoforms. The major isoform of CD22 (CD22beta) comprises 847 amino acids and seven immunoglobulin-like regions in the extracellular domain (see Wilson, G. L. et al., J. Exp. Med. 173:137-146 (1991)). A minor isoform, CD22alpha, comprises 647 amino acids and lacks immunoglobulin- like domains 3 and 4 in the extracellular domain (see Stamenkovic, I. and Seed, B., Nature 345:74-77 (1990)) and Wilson et al. (1991), supra).

[0134] The targeting component can include any type of molecule that is configured to specifically bind to cell surface CD22. Such molecules can include proteins, protein fragments, polynucleotides such as ribonucleic acids or deoxyribonucleic acids, polypeptides, polysaccharides, lipids, amino acids, peptides, sugars and / or other small or large molecules and / or polymers identifiable by skilled persons upon reading the present disclosure.

[0135] The antibody or antibody fragment described herein may reversibly bind to CD22 on the surface of a cell. This binding typically occurs at near-neutral pH, such as at a pH greater than about 6.8 and less than about 7.5, because that is the typical pH of extracellular space. CD22 with an attached antibody or antibody fragment are internalized into the cell through receptor-mediated uptake into an endosome. The complex of the antibody or antibody fragment dissociate from the CD22 in the early or late endosome due to the acidic pH (from about pH 5.0 to about pH 6.5) in these compartments. Accordingly, CD22 may recycle back in a recycling endosome to the cell surface and be reloaded with an antibody or antibody fragment, whereas the endosomally dissociated antibody or antibody fragment enters the lysosomes and is degraded into fragments. The antibody or antibody fragment is configured to bind to the cell surface CD22 under the conditions in the extracellular space, with dissociation in the endosomes or late endosomes of at least 10% of the antibody or antibody fragment that is internalized into the cell.

[0136] Endolysosomal targeting conjugates according to this disclosure are configured to specifically bind CD22 at near-neutral pH via a targeting component. The term "specifically bind" as used herein refers to a detectable selective inter-molecular interaction between the targeting component and the cell surface CD22. For example, to specifically bind, the targeting component needs to show a detectable interaction with the cell surface receptor or cell surface molecule that is being targeted, whilst not showing a detectable interaction, or much lower affinity interaction, with other cell surface receptors or cell surface molecules.Techniques for detecting specific binding to cell surface receptors are known within the art, such as flow cytometry and other methods identifiable by skilled persons.

[0137] Accordingly, endolysosomal targeting conjugates allow the linked cargo component to be internalized into cells that express the cell surface CD22 and thereafter intracellularly degraded.

[0138] Endolysosomal targeting conjugates may contain human or humanized proteins or protein fragments to avoid or decrease the possibility of an immune reaction to the endolysosomal targeting conjugates when administered to a human. The targeting component and cargo component are preferably a human protein or protein fragment for administration of the endolysosomal targeting conjugate to a human. The targeting component and cargo component are preferably a human protein or protein fragment, such as a human antibody, antibody fragment or human albumin or albumin fragment, or a humanized antibody or humanized antibody fragment for administration of the endolysosomal targeting conjugate to a human. If an endolysosomal targeting conjugate is developed for use in a non-human animal, then proteins or protein fragments derived from or engineered to be immunologically compatible with that animal may be used instead.

[0139] The terms “anti-CD22 antibody” and “an antibody that binds to CD22” refer to an antibody that is capable of binding CD22 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD22. In one embodiment, the extent of binding of an anti-CD22 antibody to an unrelated, non-CD22 protein is less than about 10% of the binding of the antibody to CD22 as measured, e.g., by ELISA. In certain embodiments, an anti-CD22 antibody binds to an epitope of CD22 that is conserved among CD22 from different species.

[0140] The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0141] The present invention provides an endolysosomal targeting conjugate, comprising a targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to cell surface CD22 of a target cell. Therefore, the term “endolysosomal targeting conjugate” encompasses antibody molecules, antibody fragments, antibody domains and nanobodies as described herein. Therefore, all subject matter relating to the antibodies or antibody fragments described herein also may apply to the endolysosomal targeting conjugates described herein. Conversely, all subject matter relatingto the endolysosomal targeting conjugates described herein also may apply to the antibodies or antibody fragments described herein.

[0142] The present invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0143] In another aspect, the present invention provides an antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

[0144] In a natural antibody molecule, there are two heavy chains and two light chains. Each heavy chain and each light chain has at its N-terminal end a variable domain. Each variable domain is composed of four framework regions (FRs) alternating with threecomplementarity determining regions (CDRs). The residues in the variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system is used in the present specification except where otherwise indicated.

[0145] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0146] The antibody fragment disclosed herein may be selected from the group consisting of antigen-binding fragments (Fab), F(ab’)2, single chain variable fragments (scFv), an antibody domain, a nanobody and “third generation” (3G) fragments.

[0147] The fragment antigen-binding region (Fab) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain.

[0148] F(ab')2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc fragment while leaving some of the hinge region intact. F(ab')2 fragments have two antigen-binding (Fab) regions linked by disulfide bonds, making them divalent.

[0149] Single-chain variable fragments (scFv) are single polypeptides that contain the variable light chain (VL) and variable heavy chain (VH) of an antibody. These two chains are connected by a flexible linker peptide that is usually 15-20 amino acids long.

[0150] “3G” fragment technologies include single domain and “miniaturized” antibody therapeutic molecules.

[0151] Single-domain antibodies, also known as nanobodies, are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

[0152] As understood by persons skilled in the art, the Fc fragment of an IgG is all of the lower base of the antibody's Y-shape, which includes a sulfhydryl-bridged hinge region and CH2 and CH3 domains. The Fc fragment may be homodimeric. Further, the Fc fragmentmay be heterodimeric, for example in bispecific or multispecific antibodies. Cargo molecules may be attached to the hinge region via chemical conjugation to cysteine residues.

[0153] Fab fragments, scFv fragments or protein fragments or domains may be linked to immunoglobulin CH3 domains to form a homodimer. Protein fragments or domains may be linked to the N-termini of an Fc fragment to form homodimers. Protein fragments or domains may be linked to the C-termini of an Fc fragment to form homodimers or to both the N- and C-termini of an Fc fragment to form homodimers.

[0154] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli, CHO or HEK293 cells).

[0155] The antibody constant region domains of an antibody of the present disclosure, if present, for example in a full length antibody or a multispecific molecule, may be selected having regard to the proposed function of the multispecific antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the lgG1 and lgG4 isotypes.

[0156] In some embodiments, the antibody or antibody fragment may comprise a heavy chain constant region selected from lgG1 , lgG2, lgG3 and lgG4, preferably lgG1 and lgG4, most preferably lgG1.

[0157] In some embodiments, the antibody or antibody fragment may comprise wherein the antibody or antibody fragment comprises a light chain constant region of kappa.

[0158] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs or CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0159] The antibody or antibody fragment of the invention may comprise: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; furthercomprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0160] The antibody or antibody fragment of the invention may comprise: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

[0161] The antibody or antibody fragment of the invention may comprise: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 12.

[0162] The antibody or antibody fragment of the invention may comprise a heavy chain comprising the amino acid sequence of a) SEQ ID NO: 13; or b) SEQ ID NO: 16.

[0163] The antibody or antibody fragment of the invention may comprise a light chain comprising the amino acid sequence of SEQ ID NO: 19.

[0164] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, I g E, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, E, y, and p, respectively.

[0165] In some embodiments, the antibody or antibody fragment may be a human antibody or antibody fragment.

[0166] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibodyencoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0167] In some embodiments, the antibody may be a full length antibody.

[0168] The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0169] In some embodiments, the antibody or antibody fragment may comprise wherein the antibody or antibody fragment is monospecific or bispecific.

[0170] “Monospecific” as employed herein refers to the ability to bind only one target antigen.

[0171] Multispecific antibodies are antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for CD22 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CD22. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD22. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

[0172] In some embodiments, the antibody or antibody fragment may comprise wherein the antibody or antibody fragment is humanised, optimised, de-immunized and / or conjugated.

[0173] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0174] “Optimised” as employed herein is intended to refer to where one, two, three, four, five or more amino acids in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is / are retained.

[0175] “De-immunized” refers to a technology for location and removal of T-cell epitopes through the combined use of immunological and molecular biology techniques. In the case of deimmunization of antibodies, mutations to remove T-cell epitopes can generally be introduced without significantly reducing the binding affinity of the antibody. Typically,"deimmunized" antibodies are created with human constant regions and by expression of genes encoding these antibodies in mammalian cells.

[0176] “Conjugated” generally means joined or bound to another molecule. The terms “conjugating,” “joining,” “bonding” or “linking” refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an scFv. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule. In one example, “conjugated” also covers wherein a component is fused directly or indirectly to another component.

[0177] Antibody variable regions may include portions of a non-variable region of an antibody that is configured to bind to a cell surface receptor or cell surface molecule. For example, antibody variable regions may be a single-domain antibody (sdAb) or camelid- derived VHH domain (also commonly referred to as a nanobody). Such variable regions have the overall fold of an immunoglobulin domain, comprising two anti- parallel p-sheets, and can also include domains from other members of the immunoglobulin superfamily such as T cell receptor variable domains, constant region domains of antibodies or domains of the coreceptor, CD4, among others identifiable by persons skilled-in-the-art. Antibody variable regions can also include heterodimers of heavy chain variable (VH) domains linked by peptide linkers to light chain variable (VL) domains to form scFv fragments. The linker sequences that are used to link VH and VL domains are well known to those with skill in the art and include the GGGGSGGGGSGGGGS [(G4S)3] sequence (SEQ ID NO: 41) that connect the C-terminus of the VH domain to the N-terminus of the VL domain. The C- terminus of the VL domain can be connected to the N-terminus of the VH domain with similar linker sequences. ScFvs that bind to a cell surface receptor or other cell surface molecule with pH-dependence can be isolated from libraries of scFvs using phage display, yeast display or other antibody display approaches. The targeting component of an endolysosomal targeting conjugate may include Fab fragments of an antibody that can be isolated from libraries of Fab fragments using phage display or yeast display among other techniques known to skilled persons. For nanobodies, scFvs and Fab fragments, the desired binding properties (pH-dependence) for the targeted cell surface receptor or cell surface molecule can be further improved by randomly mutating residues in the complementarity determining regions (CDRs), or by using error-prone polymerase chain reaction, to generate libraries of mutated nanobodies or variable domains, followed by selection. These libraries can bedisplayed on phage, yeast or mammalian cells and variants with the required binding behaviour selected using approaches known to those with skill in the art.

[0178] Antibody variable domains or fragments may be linked to albumin or an albumin fragment, which may be mutated or modified so that it binds with increased affinity to a neonatal Fc receptor (FcRn). For example, mutations can be inserted into the FcRn binding domain (Dill) of (human serum) albumin using error prone PCR followed by display of libraries of mutated albumin variants on yeast, phage or mammalian cells, and selection of higher affinity variants. Alternatively, higher affinity variants can be generated by mutating residues at or near the albumin: FcRn interface and either selecting or screening for albumin variants with increased binding affinity. Antibody variable domains or fragments may bind at a terminal location of albumin or albumin fragment, or they may instead be located at a nonterminal location. An antibody variable domain or fragment may be fused to albumin or albumin fragment in any suitable manner, including attachment via a chemical reaction or attachment through a peptide linker. The cargo molecule may be attached via chemical conjugation to an exposed amino acid such as cysteine or lysine.

[0179] In order to avoid Fc fragment homodimers in which both Fc fragments have a fused protein fragment or domain, or no fused protein or protein domain, the endolysosomal targeting conjugate can be designed with knobs-into-holes mutations (for example, as described in Moore, G.L., Bautista, C, Pong, E., Nguyen, D.H., Jacinto, J., Eivazi, A., Muchhal, U.S., Karki, S., Chu, S.Y., Lazar, G.A. (2011) A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 3, 546-557) and / or electrostatic steering mutations (for example, as described in Gunasekaran, K., Pentony, M., Shen, M., Garrett, L., Forte, C, Woodward, A., Ng, S.B., Born, T., Retter, M., Manchulenko, K., Sweet, H., Foltz, I.N., Wittekind, M., Yan, W. (2010) Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J. Biol. Chem. 285, 19637-19646) to promote heterodimer formation. Other approaches can also be used to generate heterodimers, such as the insertion of a (G4S)3 linker peptide between the C-terminus of the antigen-Fc fusion and N-terminus of a second Fc fragment (for example, as described in Zhou, L., Wang, H-Y., Tong, S., Okamoto, C.T., Shen, W-C, Zaro, J.L. (2016) Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery. Biomaterials, 117, 24-31).

[0180] Examples of knobs-into-holes mutations include Y349T / T394F: S364H / F405A and Y349T / F405F: S364H / T394F (for example, as described in Moore, G.L., Bautista, C, Pong, E., Nguyen, D.H., Jacinto, J., Eivazi, A., Muchhal, U.S., Karki, S., Chu, S.Y., Lazar, G.A. (2011) A novel bispecific antibody format enables simultaneous bivalent and monovalent coengagement of distinct target antigens. MAbs 3, 546-557) and T366W / T366S:L368A:Y407V(for example, as described in Atwell, S., Ridgway, J.B.B., Wells, J. A., Carter, P. (1997) Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol., 270, 26-35) among others identifiable by persons skilled in the art. The residue numbering of these exemplary knobs-into-holes mutations refers to the Ell antibody numbering system, as would be understood by persons skilled in the art.

[0181] Examples of electrostatic steering mutations include E356K / D399K:K392D / K409D and K409D / K370D:D357K / D399K (for example as described in Gunasekaran, K., Pentony, M., Shen, M., Garrett, L., Forte, C, Woodward, A., Ng, S.B., Born, T., Retter, M, Manchulenko, K., Sweet, H., Foltz, I.N., Wittekind, M, Yan, W. (2010) Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG. J. Biol. Chem. 285, 19637-19646) among others identifiable by persons skilled in the art. The residue numbering of these exemplary electrostatic steering mutations refers to the Ell antibody numbering system, as would be understood by persons skilled in the art.

[0182] Endolysosomal targeting conjugates may include different numbers of targeting components and cargo components.

[0183] In some examples herein described, endolysosomal targeting conjugates may include a toxin, such as monomethyl auristatin E (MMAE), conjugated via a valine- citrulline- PAB linker.

[0184] The engineered antibodies and proteins described herein can be expressed by mammalian cells at high yields and conjugated with the drug at high efficiency.

[0185] The targeting components of the endolysosomal targeting conjugates described herein that bind to a target cell surface molecule in a pH-dependent way can be isolated from libraries of immunoglobulin variable domains, scFvs (VH:VL heterodimers in which VH and VL domains are connected to each other by linker peptides such as GGGGSGGGGSGGGGS, SEQ ID NO: 41) or Fab fragments using phage display, yeast display, mammalian display or other methods identifiable by those with skill in the art. These libraries can either be derived from naturally occurring antibody variable genes, can be generated using approaches that result in 'semi -synthetic' libraries wherein complementarity determining regions (CDRs) are produced using randomized oligonucleotide sequences, or can be derived from VH and VL domain genes of existing antibodies by insertion of random mutations into the CDRs. Random mutations in the CDRs can be inserted using error-prone PCR or with biases towards histidine residues (for pH-dependence) followed by selection using phage display or yeast display. Selection of scFvs or Fab fragments with desired pH-dependence can be carried out using methods known to those with skill in the art. In addition, to isolate pH-dependent binders, CDRresidues can be systematically mutated to histidine, the resulting Fab or scFv fragments expressed and analyzed for binding to target using, for example, surface plasmon resonance or ELISAs.

[0186] The endolysosomal targeting conjugates described herein may have variations in numbers of targeting components (e.g. Fab fragments or scFv fragments) that range from 1- 4 targeting components. These targeting components may be linked to immunoglobulin Fc fragments or other proteins such as albumin, and include linker sequences that vary in length and composition between the fusion proteins, domains or fragments e.g. GGGGS (SEQ ID NO: 42) or 2-3 repeats of this linker, among other linker sequences identifiable by skilled persons. The Fc fragments of an endolysosomal targeting conjugate may also have mutations such as knobs-into-holes and / or electrostatic steering mutations so that heterodimers of Fc fragments with and without linked targeting components are formed.

[0187] The targeting component may be fused to an Fc region of an antibody, which retains the therapeutic functions and in vivo persistence elicited by the Fc region while reducing the size of the protein. In other examples, the Fc region may be replaced by albumin, or domain III of albumin, which have prolonged in vivo persistence due to the interaction of albumin (or Dili) with the recycling receptor, FcRn. In a further example, Fc mutations can be introduced to extend the half-life of the antibody or the antibody-drug conjugate. For example the YTE (M252Y / S254T / T256E) mutation and / or the LS (M428L and N434S) mutation.

[0188] The antibody or antibody fragment described herein may further have mutated amino acid residues as follows: a) position Cys220 mutated to Ser220 and Cys229 mutated to Ser229 in the heavy chain; and Cys214 mutated to Ser214 in the light chain; or b) Cys220 mutated to Ser220 in the heavy chain and Cys214 mutated to Ser214 in the light chain; according to Ell numbering. These mutated amino acid residues are located in the hinge region or at the end of the light chain constant region of the antibody or antibody fragment described herein.

[0189] The antibody or antibody fragment of described herein may comprise a heavy chain hinge region comprising the amino acid sequence of a) SEQ ID NO: 14; or b) SEQ ID NO: 17.

[0190] The antibody or antibody fragment described herein may have drug to antibody ratios (DARs) of a) two; or b) four. The term “drug to antibody ratio” (DAR) means the number of molecules of drug per molecule of antibody or antibody fragment. A specific DAR is achieved by reducing the numbers of conjugatable cysteine residues, in the amino acid sequence of the hinge region or at the end of the light chain constant region of the antibodyor antibody fragment described herein, by mutating these residues to serines, and then using standard coupling methods (e.g. Kang et al., 2019, Nature Biotech).

[0191] The antibody or antibody fragment described herein may have an FcgR-ablating mutation. “Fc gamma receptor (FcgR) binding” refers to interactions that are detectable using assays known to those skilled in the art. The antibody or antibody fragment described herein may have LALA-PG mutations. “LALA-PG mutations” refers to mutations in L234A, L235A, and P329G (Ell numbering; Schlothauer et al., 2016). These mutation sets ablate Fc gamma receptor binding i.e. they reduce or eliminate the binding of the Fc region of antibodies to Fc gamma receptors. Other examples of FcgR-ablating mutations include mutations to N297, such as N297A, N297G and N297Q; F234A / L235A; L235E; G236R / L328R; L234A / L235A / G237A; L234F / L235E / P331S and L234A / L235A / K322A mutations.

[0192] As described herein, the terms “cargo component” and “cargo molecule” refer to functional molecules bound to or conjugated to the antibody or antibody fragment of the invention or to the targeting component of the endolysosomal targeting conjugate of the invention, which have a functional use and may cause an effect in a target cell.

[0193] The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0194] Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0195] A cargo component or cargo molecule may comprise an imaging label, such as a radiolabel or a fluorescent or a near infrared label.

[0196] A cargo component or cargo molecule may comprise a linker.

[0197] “Linker” refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a drug moiety. Nonlimiting exemplary linkers are described herein. In one aspect, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0198] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, butare not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting exemplary such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0199] A linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N — Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“MCC”). Various linker components are known in the art.

[0200] A linker may be a “cleavable linker,” facilitating release of a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), proteasesensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[0201] A cargo component or cargo molecule may comprise a drug. The drug may be a cytotoxic drug. The cytotoxic drug may be maleimidocaproyl-valine-citrulline-p- aminobenzoyloxycarbonyl-monomethyl auristatin E (MMAE). The cytotoxic drug may be deruxtecan.

[0202] The exemplary endolysosomal targeting conjugate ADCs and PDCs described herein show improved efficacy in killing cancer cells through improved intracellular release of the toxin. In addition to the exemplary cytotoxic drug, MMAE, other cytotoxic drugs may be used such as a maytansinoid, tubulysin, benzodiazepine, duocarmycin, among drugs identifiable by those with skill in the art. The drug may be conjugated to the antibody, antibody fragment, antibody domain, nanobody, protein, protein fragment or protein domain of the targeting component through chemical conjugation. Examples of chemical coupling that may be used are: amine-to-amine (NHS esters), sulfhydryl-to-sulfhydryl (maleimide), amine-to-sulfhydryl (NHS ester / maleimide), sulfhydryl-to-carbohydrate (maleimide / hydrazide), or attachment via an unnatural amino acid with the desired chemical reactivity, among other approaches identifiable by skilled persons. The unnatural amino acid may be inserted during recombinant production of the targeting component. Polyethyleneglycol (PEG) spacers may be inserted between the chemically conjugated proteins, protein fragments or other molecules. Linkers may be cleavable, such as valine-citrulline to enable release of the cytotoxic drug in the late endosomes or lysosomes by resident proteases such as cathepsins. In cases where the linkage is not cleavable, such as for trastuzumab-DMI, the antibody may be proteolysed to release the drug. Linkage chemistry, sites of linkage and choice of peptide can be guided bymolecular modeling, and can be designed to minimize loss of binding activity of the ADC or PDC for cell surface receptor or other cell surface molecule, as would be understood by skilled persons.

[0203] The term “cytotoxic agent” or “cytotoxic drug” as used herein refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211 , 1131 , 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and the various antitumor or anticancer agents disclosed below.

[0204] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammal l and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5- Fll); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, III.), and docetaxel (TAXOTERE®; Rhone-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; CVP, an abbreviation for a combined therapy of cyclophosphamide, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

[0205] The cargo component of endolysosomal targeting conjugate LCs can comprise an imaging label identifiable by persons skilled in the art. Non-limiting examples of imaging labels include near infrared dyes such as IRDye800CW, or radiolabels such as 1-124, Cu-64 or Zr- 89. Conjugation to Cu-64 or Zr-89 can be achieved through chelation to 1 ,4,7, 10- tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid (DOTA) which chelates these radiolabels, among other methods identifiable by persons skilled in the art.

[0206] In further examples, the cargo component of the endolysosomal targeting conjugate can comprise a cytotoxic radiolabel (e.g. Yttrium-90, Y-90, or iodine-131 , 1-131) or drug or other agent that modifies the behavior of the targeted cell. For example, the drug could be an antagonistic ligand for the androgen receptor (AR) and could be used to downregulate AR activity.

[0207] The endolysosomal targeting conjugates herein described may be provided in a composition that includes an endolysosomal targeting conjugate and a pharmaceutically acceptable vehicle.

[0208] The present invention provides a pharmaceutical composition comprising the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, and / or the host cell of the invention; and further comprising a pharmaceutically acceptable carrier.

[0209] “Pharmaceutically acceptable carriers” or “pharmaceutically acceptable vehicles” are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs andmethods of use, including rHuPH20, are described in US Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0210] The present invention provides a nucleic acid encoding the antibody or antibody fragment of the invention.

[0211] In one embodiment, the antibody or antibody fragment of the invention comprises a heavy chain with the nucleic acid sequence of SEQ ID NO: 15.

[0212] In another embodiment, the antibody or antibody fragment of the invention comprises a heavy chain with the nucleic acid sequence of SEQ ID NO: 18.

[0213] In one embodiment, the antibody or antibody fragment of the invention comprises a light chain with the nucleic acid sequence of SEQ ID NO: 20.

[0214] A “nucleic acid” as described herein (or “isolated nucleic acid”) refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0215] The present invention provides an expression vector comprising the nucleic acid of the invention.

[0216] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0217] The present invention provides a host cell comprising the expression vector of the invention.

[0218] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0219] The endolysosomal targeting conjugates or compositions thereof described herein may be administered using any suitable method to deliver them to a subject, such as a cell, a plurality of cells, or a multicellular organism, in particular an animal or a human, and in particular an animal or a human that may have one or more tumours, such as via injection, particularly intravenous, subcutaneous or intramuscular injection, among other methods identifiable by skilled persons.

[0220] Any of the endolysosomal targeting conjugates or compositions thereof described herein or the anti-CD22 antibodies or antibody fragments described herein may be used in methods, e.g., therapeutic methods.

[0221] As used herein the terms “treat”, “treating” or “treatment” refer to a clinical improvement of for example cancer in a subject with this disease or preventing the development of a benign tumour to a malignant tumour. A clinical improvement may be demonstrated by an improvement of the pathology and / or symptoms associated with the cancer.

[0222] In some embodiment, effective treatment may be demonstrated by slowing or halting the progression of the disease in the subject, or reversing the disease. Suitably, the disease may be reversed partially, or completely. In some embodiments, complete reversal of the diseases may be sufficient to result in curing of the disease.

[0223] A clinical improvement may be demonstrated by an improvement of the pathology and / or symptoms associated with the cancer or pre-cancer. Suitably, therapeutic effect (or “a therapeutically effective amount”) may be demonstrated by preventing the development of the cancer or pre-cancer in a subject, slowing or halting the progression of the cancer or precancer in the subject, or reversing the cancer or pre-cancer. Suitably, the cancer or pre- cancer may be reversed partially, or completely. Clinical improvement of the pathology may be demonstrated by one or more of the following: reduced cancer or pre-cancer biomarker levels in the subject, reduced cancer or pre-cancer cell number in the subject, increased time to regrowth of cancer upon stopping of treatment, prevention or delay of pre-cancer development into cancer, prevention of regrowth of cancer upon stopping treatment, decreased tumour invasiveness, reduction or complete elimination of metastasis, increased cancer cell differentiation, or increased survival rate. In some embodiments, anti-tumour effects may be demonstrated by inhibition of tumour growth, reduced speed of tumour growth, or a partial or complete reduction in tumour mass / lump. Other suitable indications of clinical improvement in the pathology will be known to the skilled person. It will be appreciated that indications of clinical improvement of the pathology will vary depending on the type of cancer. Clinical improvement of symptoms associated with cancer may be, butare not limited to, partial or complete alleviation of pain and / or swelling, increased appetite, reduced weight loss, and / or reduced fatigue.

[0224] Suitably, in the methods of the invention, the endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be administered in a therapeutically effective amount. The term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in a clinical improvement of cancer.

[0225] In the context of the present disclosure, the term “subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In suitable embodiments, subjects are mammals, particularly primates, especially humans. In suitable embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In certain embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like. Herein, the terms, “patients” and “subjects” may be used interchangeably.

[0226] The endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be used in a method of treating a subject.

[0227] The invention provides the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in therapy.

[0228] The endolysosomal targeting conjugate or composition thereof of the invention may be for use in therapy.

[0229] The endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be used in a method of treating cancer.

[0230] The invention provides the antibody or antibody fragment of the invention, the nucleic acid of the invention, the expression vector of the invention, the host cell of the invention and / or the pharmaceutical composition of the invention for use in treating cancer.

[0231] The endolysosomal targeting conjugate or composition thereof of the invention may be for use in treating cancer.

[0232] The method of treating cancer includes administering a subject with an effective dose of the endolysosomal targeting conjugate or a composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition to a subject, wherein the cargo molecule is a cytotoxic drug and the administering suppresses growth of a tumour in the subject.

[0233] The cancer may be a tumour expressing CD22. The cancer may be a B cell tumour expressing CD22. The cancer may be selected from the group consisting of: B-cell malignancies, B cell acute lymphoblastic leukaemia, non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B cell lymphoma.

[0234] B cell-related disorders include, but are not limited to, malignant lymphoma (NonHodgkin's Lymphoma, NHL), multiple myeloma, and chronic lymphocytic leukemia (CLL, B cell leukemia (CD5+ B lymphocytes). Non-Hodgkin's lymphomas (NHLs), a heterogeneous group of cancers principally arising from B lymphocytes, represent approximately 4% of all newly diagnosed cancers (Jemal, A. et al., CA-Cancer J Clin, 52: 23-47, (2002)). Aggressive NHL comprises approximately 30-40% of adult NHL (Harris, N. L. et al., Hematol. J. 1 :53-66 (2001)) and includes diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), peripheral T-cell lymphoma, and anaplastic large cell lymphoma. Frontline combination chemotherapy cures less than half of the patients with aggressive NHL, and most patients eventually succumb to their disease (Fisher, R. I. Semin. Oncol. 27(suppl 12): 2-8 (2000)).

[0235] In B-cell NHL, CD22 expression ranges from 91% to 99% in the aggressive and indolent populations, respectively (Cesano, A. et al., Blood 100:350a (2002)). CD22 may function both as a component of the B-cell activation complex (Sato, S. et al., Semin. Immunol. 10:287-296 (1998)) and as an adhesion molecule (Engel, PI t al., J. Immunol. 150:4719-4732 (1993)). The B cells of CD22-deficient mice have a shorter life span and enhanced apoptosis, which suggests a role of this antigen in B-cell survival (Otipoby, K. L. et al., Nature (Lond) 384:634-637 (1996)). After binding with its natural ligand(s) or antibodies, CD22 is rapidly internalized, providing a costimulatory signal in primary B cells and proapoptotic signals in neoplastic B cells (Sato, S. et al., Immunity 5:551-562 (1996)).

[0236] Examples of cancer include, but are not limited to, melanoma, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of cancer include B-cell associated cancers, including for example, high, intermediate and low grade lymphomas (including B cell lymphomas such as, for example, mucosa-associated-lymphoid tissue B cell lymphoma and non-Hodgkin's lymphoma (NHL), mantle cell lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, diffuse large cell lymphoma, follicular lymphoma, and Hodgkin'slymphoma and T cell lymphomas) and leukemias (including secondary leukemia, chronic lymphocytic leukemia (CLL), such as B cell leukemia (CD5+ B lymphocytes), myeloid leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, lymphoid leukemia, such as acute lymphoblastic leukemia (ALL) and myelodysplasia), and other hematological and / or B cell- or T-cell-associated cancers. Also included are cancers of additional hematopoietic cells, including polymorphonuclear leukocytes, such as basophils, eosinophils, neutrophils and monocytes, dendritic cells, platelets, erythrocytes and natural killer cells. Also included are cancerous B cell proliferative disorders selected from the following: lymphoma, non-Hodgkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), and mantle cell lymphoma. The origins of B-cell cancers include as follows: marginal zone B-cell lymphoma origins in memory B-cells in marginal zone, follicular lymphoma and diffuse large B-cell lymphoma originates in centrocytes in the light zone of germinal centers, chronic lymphocytic leukemia and small lymphocytic leukemia originates in B1 cells (CD5+), mantle cell lymphoma originates in naive B-cells in the mantle zone and Burkitt's lymphoma originates in centroblasts in the dark zone of germinal centers. Tissues which include hematopoietic cells referred herein to as “hematopoietic cell tissues” include thymus and bone marrow and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa, such as the gut-associated lymphoid tissues, tonsils, Peyer's patches and appendix and lymphoid tissues associated with other mucosa, for example, the bronchial linings. Further particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0237] A “B-cell malignancy” herein includes non-Hodgkin's lymphoma (NHL), including low grade / follicular NHL, small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia, non-Hodgkin's lymphoma (NHL), lymphocyte predominant Hodgkin's disease (LPHD), small lymphocytic lymphoma(SLL), chronic lymphocytic leukemia (CLL), indolent NHL including relapsed indolent NHL and rituximab-refractory indolent NHL; leukemia, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia, chronic myeloblastic leukemia; Burkitt's lymphoma; mantle cell lymphoma; and other hematologic malignancies. Such malignancies may be treated with antibodies directed against B-cell surface markers, such as CD22. Such diseases are contemplated herein to be treated by the administration of an antibody directed against a B cell surface marker, such as CD22, and includes the administration of an unconjugated (“naked”) antibody or an antibody conjugated to a cytotoxic agent as disclosed herein. Such diseases are also contemplated herein to be treated by combination therapy including an anti-CD22 antibody or anti-CD22 antibody drug conjugate of the invention in combination with another antibody or antibody drug conjugate, another cytoxic agent, radiation or other treatment administered simultaneously or in series.

[0238] The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to a cancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin's lymphomas can generally be distinguished from non-Hodgkin's lymphomas by the presence of Reed-Sternberg cells in Hodgkin's lymphomas and the absence of said cells in non-Hodgkin's lymphomas. Examples of non-Hodgkin's lymphomas encompassed by the term as used herein include any that would be identified as such by one skilled in the art (e.g., an oncologist or pathologist) in accordance with classification schemes known in the art, such as the Revised European- American Lymphoma (REAL) scheme as described in Color Atlas of Clinical Hematology (3rd edition), A. Victor Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Ltd., 2000). See, in particular, the lists in FIGS. 11.57, 11.58 and 11.59. More specific examples include, but are not limited to, relapsed or refractory NHL, front line low grade NHL, Stage lll / IV NHL, chemotherapy resistant NHL, precursor B lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B cell chronic lymphocytic leukemia and / or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone — MALT lymphoma, nodal marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasma cell myeloma, low grade / follicular lymphoma, intermediate grade / follicular NHL, mantle cell lymphoma, follicle center lymphoma (follicular), intermediate grade diffuse NHL, diffuse large B-cell lymphoma, aggressive NHL (including aggressive front-line NHL and aggressive relapsed NHL), NHL relapsing after or refractory to autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary effusion lymphoma, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor (peripheral) large granular lymphocyticleukemia, mycosis fungoides and / or Sezary syndrome, skin (cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentric lymphoma.

[0239] The endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be used in a method of imaging a tumour in a subject. The endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be for use in imaging a tumour in a subject. The method includes the steps of: administering a subject with an effective dose of the endolysosomal targeting conjugate or a composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition to a subject, wherein the cargo molecule is an imaging label; and performing an imaging method suitable for detecting the imaging label in the subject. In the method, the administering is performed at an effective dose to provide a sufficient concentration of the imaging label that is detectable by the imaging method, as would be identifiable by skilled persons.

[0240] In a further embodiment, an anti-CD22 antibody is used in vivo to detect, e.g., by in vivo imaging, a CD22-positive cancer in a subject, e.g., for the purposes of diagnosing, prognosing, or staging cancer, determining the appropriate course of therapy, or monitoring response of a cancer to therapy. One method known in the art for in vivo detection is immunopositron emission tomography (immuno-PET), as described, e.g., in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel et al., J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method is provided for detecting a CD22-positive cancer in a subject, the method comprising administering a labeled anti-CD22 antibody to a subject having or suspected of having a CD22-positive cancer, and detecting the labeled anti-CD22 antibody in the subject, wherein detection of the labeled anti-CD22 antibody indicates a CD22-positive cancer in the subject. In certain of such embodiments, the labeled anti-CD22 antibody comprises an anti-CD22 antibody conjugated to a positron emitter, such as 68Ga, 18F, 64Cu, 86Y, 76Br, 89Zr, and 1241. In a particular embodiment, the positron emitter is 89Zr.

[0241] In certain embodiments, any of the anti-CD22 antibodies provided herein is useful for detecting the presence of CD22 in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. A “biological sample” comprises, e.g., a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous lymph tissue, including tissue from subjects having or suspected of having a B cell disorder and / or a B cell proliferative disorder, including, but not limited to, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractoryindolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma, and mantle cell lymphoma.

[0242] In one embodiment, an anti-CD22 antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of CD22 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-CD22 antibody as described herein under conditions permissive for binding of the anti-CD22 antibody to CD22, and detecting whether a complex is formed between the anti-CD22 antibody and CD22 in the biological sample. Such method may be an in vitro or in vivo method. In one embodiment, an anti-CD22 antibody is used to select subjects eligible for therapy with an anti-CD22 antibody, e.g. where CD22 is a biomarker for selection of patients. In a further embodiment, the biological sample is a cell or tissue (e.g., cancerous or potentially cancerous lymph tissue, including tissue of subjects having or suspected of having a B cell disorder and / or a B cell proliferative disorder, including, but not limited to, lymphoma, non-Hogkins lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), Burkitt's lymphoma, and mantle cell lymphoma.

[0243] An endolysosomal targeting conjugate, antibody or antibody fragment of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0244] An endolysosomal targeting conjugate, antibody or antibody fragment of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The endolysosomal targeting conjugate, antibody or antibody fragment need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of endolysosomal targetingconjugate, antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0245] For the prevention or treatment of disease, the appropriate dosage of an endolysosomal targeting conjugate, antibody or antibody fragment of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of endolysosomal targeting conjugate, antibody or antibody fragment, the severity and course of the disease, whether the endolysosomal targeting conjugate, antibody or antibody fragment is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the endolysosomal targeting conjugate, antibody or antibody fragment, and the discretion of the attending physician. The endolysosomal targeting conjugate, antibody or antibody fragment is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g. 0.1 mg / kg-10 mg / kg) of endolysosomal targeting conjugate, antibody or antibody fragment can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the endolysosomal targeting conjugate, antibody or antibody fragment would be in the range from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0246] The endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be administered at suitable time intervals, for example weekly, monthly or, for example, whenever 50% of subjects are expected to have shown tumour regression.

[0247] The endolysosomal targeting conjugate, antibody or antibody fragment of the invention can be used either alone or in combination with other agents in a therapy. For instance, an endolysosomal targeting conjugate, antibody or antibody fragment of the invention may be co-administered with at least one additional therapeutic agent.

[0248] In diagnostic / theranostic imaging, it is expected that the administration to a subject of an endolysosomal targeting conjugate LC or an antibody or antibody fragment, nucleic acid, expression vector, host cell and / or pharmaceutical composition that includes an imaging label such as a radiolabel, near infrared label, or fluorescent label may be followed by a period of 1-7 days to allow localization of the target cell in the subject, for example, tumour localization in the subject. Following this period, the subject may be imaged using positron emission tomography or other suitable imaging modality, such as localized or whole-body imaging, to allow detection of the location of the target cell such as the tumour.

[0249] The endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition described herein may be designed to selectively target a particular cell type, and thereby deliver a cargo molecule to a selected target cell. In particular, the endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition described herein may be designed to target a particular type of tumour cell in a subject. Accordingly, a method of providing the endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition described herein may include the steps of: (1) selecting a targeting component, wherein the targeting component includes an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain configured to selectively bind to a cell surface molecule on a selected type of cell, such as a tumour target cell, wherein the targeting component is configured to bind to a cell surface molecule that is expressed on the target cell with higher affinity in an extracellular space than in an endolysosomal compartment; (2) selecting a cargo component, comprising a cargo molecule, wherein the cargo molecule may be, for example, a cytotoxic drug having efficacy for suppressing growth of the selected type of tumour target cell or an imaging label suitable for imaging of the selected tumour cell; and (3) providing the endolysosomal targeting conjugate or composition thereof, the antibody or antibody fragment, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition including the targeting component fused directly or indirectly to the cargo component.

[0250] The term "behaviour" in relation to a target cell or other cell may refer to an activity, a function, an output, or any other attribute or action regarding the phenotype or genotype ofthe target cell or other cell. In general, a drug or other agent can be used to produce an effect, such as a particular therapeutic effect, a cytotoxic effect, and so on, with regard to a target cell, as would be understood by skilled persons upon reading the present disclosure.

[0251] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the nucleotide bases or residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity or similarity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted with a functionally equivalent residue of the amino acid residues with similar physiochemical properties and therefore do not change the functional properties of the molecule.

[0252] A functionally equivalent residue of an amino acid used herein typically refers to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine is considered to be a functionally equivalent residue to lysine.

[0253] A person skilled in the art would understand that similarity between sequences is typically measured by a process that includes the steps of aligning the two polypeptide or polynucleotide sequences to form aligned sequences, then detecting the number of matched characters, i.e. characters similar or identical between the two aligned sequences, and calculating the total number of matched characters divided by the total number of aligned characters in each polypeptide or polynucleotide sequence, including gaps. The similarity result is expressed as a percentage of identity.

[0254] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (gaps) as compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0255] As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length protein or protein fragment. A reference sequence can be, for example, a sequence identifiable in a database such as GenBank and UniProt and others identifiable to those skilled in the art.

[0256] As understood by those skilled in the art, determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Computer implementations of suitable mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL, ALIGN, GAP, BESTFIT, BLAST, FASTA, among others identifiable by skilled persons.

[0257] For example, endolysosomal targeting conjugates, antibodies or antibody fragments according to the present disclosure may include an amino acid sequence having at least 50% sequence identity, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity compared to SEQ ID NOs 1-14, 16, 17, 19, 21 , 23 and / or 25.

[0258] For example, endolysosomal targeting conjugates, antibodies or antibody fragments according to the present disclosure may include a nucleotide sequence having at least 50% sequence identity, preferably at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity compared to SEQ ID NOs 15, 18 and / or 20.

[0259] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagentsdescribed, as these may vary, depending upon the context they are used by those of skill in the art.

[0260] WO / 2018 / 136455 is hereby incorporated by this reference in its entirety.

[0261] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0262] Aspects of the invention are demonstrated by the following non-limiting examples.EXAMPLESExample 1Generation of a mutated CD22-specific antibody with highly pH-dependent binding

[0263] A mutated CD22-specific antibody (VH and VL domain sequences derived from inotuzumab; mutants A, B and C) with increased pH-dependent binding (higher affinity at pH 7.4 than at pH 6.0) has resulted from the inventors’ randomisation of complementarity determining region (CDR) residues and panning using phage display, combined with histidine exchange of CDR residues. The dissociation constants (KDS) of the mutated variant and type (WT) inotuzumab for binding to recombinant CD22 as Fab fragments have been determined using surface plasmon resonance (SPR; BIAcore). In this context, the WT parent antibody has a higher affinity at pH 6.0 than at pH 7.4 for binding to CD22 i.e. ‘reverse’ pH- dependence to that required (Table 1; data for binding of Fab fragments is shown). Mutant A has a substantial increase in pH-dependent binding, and also has a ~4-fold higher affinity at pH 7.4 compared with the WT parent antibody (Table 1). By contrast, Mutants B and C have lower affinities and do not have as marked pH-dependence as Mutant A (Table 1).

[0264] WT inotuzumab and mutants A, B and C have been expressed as full length human lgG4 [with stabilizing hinge mutation to ablate Fab-arm exchange; (1,2)] and human lgG1 antibodies and purified. Binding of human lgG1-based ADCs to FcyRs has been suggestedto lead to off-target toxicities due to uptake by FcyR-expressing cells (3), and the inventors have therefore ablated binding to FcyRs in the human IgG 1 constructs by inserting LALA-PG mutations (4) (other FcyR-ablating mutations could be used to achieve the same effect). The FcyR-ablated, human IgG 1 antibodies have been used in subsequent studies described below.

[0265] Determination of equilibrium dissociation constants (KDs) using surface plasmon resonance (SPR; BIAcore) of the interactions of wild type (WT) antibody and mutants A, B and C as Fab fragments with immobilised extracellular domain of human CD22 (Table 1). Fab fragments were diluted in PBS-T buffer (PBS containing 0.01% Tween-20) with pH adjusted to either pH 7.4 or pH 6.0 at concentrations ranging from 5-2560 nM (0.005-2.56 pM) and injected at a flow rate of 5 pl / minute. The equilibrium dissociation constants (KDs) at pH 7.4 or pH 6.0 were determined. The CD22-coupled flow cell of the chip was regenerated between runs by injecting 50 mM borate, pH 11 , 0.5 M sodium chloride for 60 seconds at a flow rate of 30 pl / minute.

[0266] The WT antibody and mutants A, B and C (human IgG 1 with LALA-PG mutations) have been conjugated via cysteine residues to maleimidocaproyl-valine-citrulline-p- aminobenzoyloxycarbonyl-monomethyl auristatin E (MMAE) with drug-to-antibody ratios (DARs) of two and four. These DARs were achieved by mutating the appropriate numbers of cysteines in the light and heavy chains of the antibodies to serines (5). Importantly, the MMAE-conjugated ADCs (WT and mutants A, B and C) have similar binding properties to those of the corresponding unconjugated antibodies, indicating that conjugation has not affected their interactions with CD22.Example 2Cellular analyses of an acid-switched antibody / ADC that targets CD22Materials and Methods

[0267] Accumulation of WT-MMAE and Mut A-MMAE in CD22-expressing tumour cell lines. Cells were treated with 30 pg / ml Alexa 488-labelled WT-MMAE or Mut A-MMAE (DAR=2) in growth medium for 0.5, 4 and 18 hours at 37°C in a 5% CO2 incubator. The cells were cooled on ice and incubated with 25 pg / ml Alexa Fluor 488-specific antibody for 30 minutes to quench the Alexa Fluor 488 signal associated with surface-bound ADC. The cells were then washed and analysed using a Cytoflex-S flow cytometer (Beckman Coulter). The quenching efficiency was determined using separate cell samples which were incubated withAlexa 488-labelled antibodies on ice for 30 minutes followed by treatment with Alexa Fluor 488-specific quenching antibody. Mean values for triplicate samples are shown. Error bars indicate SD, and statistically significant differences are shown (two-way ANOVA, Bonferroni correction for multiple comparisons; *p < 0.05, **p < 0.01 , ***p < 0.001).Results and DiscussionThe inventors have used flow cytometric assays to compare the subcellular accumulation of mutant A as a MMAE-conjugated ADC (Mut A-MMAE) with WT antibody (MMAE-conjugated; WT-MMAE) in the CD22-expressing cell lines Raji, Bjab (both CD22hi), and Ramos (CD22int) and in a further CD22-expressing cell line. The DAR for the ADCs used in the accumulation studies was designed to be two. The data shown (Figure 1) indicate that Mut A-MMAE accumulates to substantially higher levels than WT-MMAE in all cell lines.Materials and Methods

[0268] Internalisation of WT-MMAE and Mut A-MMAE (DAR = 2) and analysis of lysosomal delivery in CD22-expressing tumour cell lines. Cells were pulsed and chased with Alexa Fluor 647-labelled dextran (0.25 mg / ml) for 2 and 3 hours respectively (37°C, 5% CO2), followed by treatment with 0.03 mg / ml Alexa Fluor 555-labelled WT-MMAE or Mut A-MMAE for 4 and 18 hours as indicated (37°C, 5% CO2). Cells were then washed and fixed.Fluorescence images were acquired using a Zeiss Axio Observer 7 fluorescence microscope (Objective: 100x 1.4NA Zeiss. Camera: Hamamatsu ORCA Flash 4.0 V3. Light source: Xylis-X-Cite LED illuminator XT720S. Filter Sets: Semrock Brightline; Alexa Fluor 555: FF562; Alexa Fluor 647: FF660). Astero Lumio software (Astero Technologies LLC) was used with piecewise linear adjustment for image analysis. Scale bars represent 1 pm.Results and Discussion

[0269] Microscopy analyses also show that Mut A-MMAE traffics to lysosomes more efficiently than WT-MMAE (Figure 2).Materials and Methods

[0270] Cytotoxicity of ADCs comprising WT antibody and mutant A (Mut A) towards CD22- expressing tumour cells. Cells were treated with ADCs conjugated with MMAE with a DAR of two (2C) (A), four (4C) (B) or Deruxtecan (DXd) with a DAR of two (2C) (C) or four (4C) (D). As a control, a MMAE-conjugated ADC (DAR = 2) comprising a hen egg lysozyme-specific antibody (HuLyslO) was used. Following incubation for 72 hours, cell viability wasdetermined using an MTS assay. Data shown represent means of optical density (OD; 490 nm). Two-way ANOVA with Bonferroni correction for multiple comparison was used for statistical analyses, and statistical significance is shown for Mut A vs. corresponding WT ADCs (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).Results and DiscussionIn addition to conjugating WT antibody and mutant A to MMAE, these antibodies have been conjugated to the cytotoxic drug deruxtecan (WT-DXd and Mut A-DXd) and compared with the corresponding WT ADCs in cytotoxicity assays (Figure 3). For these experiments, ADCs with DARs of two (labelled ‘2C’) or four (labelled ‘4C’) were used. For both MMAE- and deruxtecan-conjugated ADCs, ADCs comprising mutant A have higher cytotoxicity than the corresponding ADCs comprising WT antibody. As expected, the deruxtecan-based ADCs have lower cytotoxicity than the corresponding MMAE-conjugated ones, due to the lower cytotoxic potency of deruxtecan.Example 3Therapy studies in miceMaterials and Methods

[0271] To compare the therapeutic effects of WT-MMAE and Mut A-MMAE (DAR = 2), mice bearing CD22-expressing tumour xenografts were treated with 4 mg / kg ADC or vehicle control at weekly intervals as indicated in Figure 4. These studies demonstrate that Mut A- MMAE is more effective in reducing tumour growth than WT-MMAE.Results and Discussion

[0272] Female SCID mice bearing CD22-expressing tumours were treated weekly (three times; arrowheads) with 4 mg / kg ADC [WT-MMAE (2C) or Mut A-MMAE (2C)] or vehicle (PBS) (n= 5-7 mice per group). The mean tumour volume for each treatment group is shown and error bars indicate SEM (A). Statistically significant differences at the experimental endpoints are indicated (two-tailed Mann-Whitney ll-test and unpaired two-tailed t-test; **p < 0.01 , ***p < 0.001). The tumour volume for each treatment group with individual mice is shown (B).Example 4Cellular analyses of an acid-switched antibody / ADC that targets CD22Materials and Methods

[0273] Accumulation of WT-MMAE, Mut A-MMAE, Mut B-MMAE or Mut C-MMAE in CD22- expressing tumour cell lines. Cells were treated with 30 pg / ml Alexa 488-labelled WT- MMAE, Mut A-MMAE, Mut B-MMAE or Mut C-MMAE (DAR=2) in growth medium for 4 and 18 hours at 37°C in a 5% CO2 incubator. The cells were cooled on ice and incubated with 25 pg / ml Alexa Fluor 488-specific antibody for 30 minutes to quench the Alexa Fluor 488 signal associated with surface-bound ADC. The cells were then washed and analysed using a Cytoflex-S flow cytometer (Beckman Coulter). The quenching efficiency was determined using separate cell samples which were incubated with Alexa 488-labelled antibodies on ice for 30 minutes followed by treatment with Alexa Fluor 488-specific quenching antibody. Mean values for triplicate samples are shown. Error bars indicate SD, and statistically significant differences are shown (two-way ANOVA, Bonferroni correction for multiple comparisons; **p < 0.01, ***p < 0.001).Results and DiscussionThe inventors have used flow cytometric assays to compare the subcellular accumulation of mutants A, B and C as a MMAE-conjugated ADCs (Mut A-MMAE, Mut B-MMAE, Mut C- MMAE) with WT antibody (MMAE-conjugated; WT-MMAE) in the CD22-expressing cell lines Raji, Bjab (both CD22hi), and Ramos (CD22int). The DAR for the ADCs used in the accumulation studies was designed to be two. The data shown (Figure 5) indicate that Mut B-MMAE and Mut C-MMAE accumulate at lower levels compared to Mut A-MMAE, which exhibits significantly higher accumulation than WT-MMAE across all cell lines. The inventors then proceeded to compare Mut A-MMAE accumulation with WT-MMAE across a broader panel of cell lines with varying CD22 surface expression levels; Raji, Bjab and Daudi (all CD22hi), Ramos and SU-DHL-4 (both CD22int) and Reh and DOHH-2 (both CD22low) (Figure 6A). In addition to conjugating WT antibody and mutant A to MMAE, these antibodies have been conjugated to the cytotoxic drug deruxtecan (WT-DXd and Mut A-DXd) and compared with the corresponding WT ADCs in cell accumulation (Figure 6B). Mut A-DXd shows higher accumulation than WT-DXd.Materials and Methods

[0274] Internalisation of WT-MMAE and Mut A-MMAE (DAR = 2) and analysis of lysosomal delivery in CD22-expressing tumour cell lines. Cells were pulsed and chased with Alexa Fluor 647-labelled dextran (0.25 mg / ml) for 2 and 3 hours respectively (37°C, 5% CO2), followed by treatment with 30 pg / ml Alexa Fluor 555-labelled WT-MMAE or Mut A-MMAE for 4 and 18 hours as indicated (37°C, 5% CO2). Cells were then washed and fixed.Fluorescence images were acquired using a Zeiss Axio Observer 7 fluorescence microscope(Objective: 100x 1.4NA Zeiss. Camera: Hamamatsu ORCA Flash 4.0 V3. Light source: Xylis-X-Cite LED illuminator XT720S. Filter Sets: Semrock Brightline; Alexa Fluor 555: FF562; Alexa Fluor 647: FF660). Astero Lumio software (Astero Technologies LLC) was used with piecewise linear adjustment for image analysis. Scale bars represent 1 pm.Results and Discussion

[0275] Microscopy analyses also show that Mut A-MMAE traffics to lysosomes more efficiently than WT-MMAE (Figure 7).Materials and Methods

[0276] Cytotoxicity of ADCs comprising WT antibody and mutant A (Mut A) towards CD22- expressing tumour cells. Cells were treated with ADCs conjugated with MMAE with a DAR of two (2C) (A) or conjugated with Deruxtecan (DXd) with a DAR of two (2C) (B). Following incubation for 72 hours, cell viability was determined using an MTS assay. Data shown represent means of optical density (OD; 490 nm). Two-way ANOVA with Bonferroni correction for multiple comparison was used for statistical analyses, and statistical significance is shown for Mut A vs. corresponding WT ADCs (*p < 0.05, **p < 0.01 , ***p < 0.001 ,Results and DiscussionAssays to compare the cytotoxicity of WT-MMAE with Mut A-MMAE and WT-DXd with Mut A-DXd have also been carried out (Figure 8). For these experiments, ADCs with DARs of two (labelled ‘2C’) were used. For both MMAE- and deruxtecan-conjugated ADCs, ADCs comprising mutant A have higher cytotoxicity than the corresponding ADCs comprising WT antibody.

[0277] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0278] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0279] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0280] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.SequencesTable 2References1. van der Neut Kolfschoten M, Schuurman J, Losen M, et al. Anti-inflammatory activity of human lgG4 antibodies by dynamic Fab arm exchange. Science 2007; 317(5844): 1554-7.2. Labrijn AF, Buijsse AO, van den Bremer ET, et al. Therapeutic lgG4 antibodies engage in Fab-arm exchange with endogenous human lgG4 in vivo. Nat Biotechnol 2009; 27(8): 767-71. 3. de Goeij BE, Lambert JM. New developments for antibody-drug conjugate-based therapeutic approaches. Current Opinion in Immunology 2016; 40: 14-23.4. Schlothauer T, Herter S, Koller CF, et al. Novel human IgG 1 and lgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Eng Des Se / 2016; 29(10): 457-66. 5. Kang JC, Sun W, Khare P, et al. Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy. Nat Biotechnol 2019; 37(5): 523-6.

Claims

Claims1. An antibody or antibody fragment that binds to human CD22, comprising: a) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 2, and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO: 3; and b) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (VLCDR2) amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (VLCDR3) amino acid sequence of SEQ ID NO: 6, further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VHCDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VLCDR3); iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VLCDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VLCDR3).

2. The antibody or antibody fragment of claim 1 comprising: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 9; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 10; further comprising at least one of the following mutations: i) Asparagine (N) to Histidine (H) in position 5 of the heavy chain complementarity determining region 3 (VH CDR3); ii) Glutamine (Q) to Histidine (H) in position 6 of the light chain complementarity determining region 3 (VL CDR3);iii) Tyrosine (Y) to Valine (V) in position 8 of the light chain complementarity determining region 3 (VL CDR3); and / or iv) Threonine (T) to Valine (V) in position 9 of the light chain complementarity determining region 3 (VL CDR3).

3. The antibody or antibody fragment of claim 1 or claim 2 comprising: a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11 ; and b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 12.

4. The antibody or antibody fragment of any of claims 1-3 wherein the antibody or antibody fragment further has mutated amino acid residues as follows: a) position Cys220 mutated to Ser220 and Cys229 mutated to Ser229 in the heavy chain; and Cys214 mutated to Ser214 in the light chain; or b) Cys220 mutated to Ser220 in the heavy chain and Cys214 mutated to Ser214 in the light chain; according to EU numbering.

5. The antibody or antibody fragment of any of claims 1-4 with drug to antibody ratios (DARs) of a) two; or b) four.

6. The antibody or antibody fragment of any of claims 1-5 comprising a heavy chain hinge region comprising the amino acid sequence of a) SEQ ID NO: 14; or b) SEQ ID NO: 17.

7. The antibody or antibody fragment of any of claims 1-6 comprising a heavy chain comprising the amino acid sequence of a) SEQ ID NO: 13; or b) SEQ ID NO: 16.

8. The antibody or antibody fragment of any of claims 1-7 comprising a light chain comprising the amino acid sequence of SEQ ID NO: 19.

9. The antibody fragment of any of claims 1-8, wherein the fragment is selected from the group consisting of antigen-binding fragments (Fab), F(ab’)2, single chain variable fragments (scFv), an antibody domain, a nanobody and “third generation” (3G) fragments.

10. The antibody or antibody fragment of any of claims 1-9, wherein the antibody or antibody fragment is a human antibody or antibody fragment.

11. The antibody of any of claims 1-10, wherein the antibody is a full length antibody.

12. The antibody or antibody fragment of any of claims 1-11, wherein the antibody or antibody fragment comprises a heavy chain constant region selected from lgG1, lgG2, lgG3 and lgG4, preferably lgG1 and lgG4, most preferably lgG1.

13. The antibody or antibody fragment of any of claims 1-12, wherein the antibody or antibody fragment comprises a light chain constant region of kappa.

14. The antibody or antibody fragment of any of claims 1-13, wherein the antibody or antibody fragment is monospecific.

15. The antibody or antibody fragment of any of claims 1-14, wherein the antibody or antibody fragment is bispecific.

16. The antibody or antibody fragment of any of claims 1-15, wherein the antibody or antibody fragment is humanised, optimised, de-immunized and / or conjugated.

17. The antibody or antibody fragment of any of claims 1-16, wherein the antibody or antibody fragment is conjugated to a cargo molecule.

18. The antibody or antibody fragment of claim 17 wherein the cargo molecule is an imaging label.

19. The antibody or antibody fragment of claim 18 wherein the imaging label is a radiolabel or a fluorescent or a near infrared label.

20. The antibody or antibody fragment of claim 17 wherein the cargo molecule is a drug and / or a linker.

21. The antibody or antibody fragment of claim 20 wherein the drug is a cytotoxic drug.

22. The antibody or antibody fragment of claim 21 wherein the cytotoxic drug is maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl-monomethyl auristatin E (MMAE).

23. The antibody or antibody fragment of claim 21 wherein the cytotoxic drug is deruxtecan.

24. An endolysosomal targeting conjugate, comprising: a) a targeting component comprising an antibody, an antibody fragment, an antibody domain, a nanobody, a protein, a protein fragment, or a protein domain, wherein the targeting component is configured to bind to cell surface CD22 of a target cell with a lower dissociation constant in an extracellular space than in an endolysosomal compartment of the target cell; and b) a cargo component comprising a cargo molecule; wherein the targeting component is fused directly or indirectly to the cargo component; wherein upon entry to the endolysosomal compartment, the targeting component is configured to dissociate from the cell surface CD22; and wherein the endolysosomal targeting conjugate is configured to deliver the cargo molecule to the endolysosomal compartment of the target cell.

25. The endolysosomal targeting conjugate of claim 24 wherein the targeting component is configured to bind to cell surface CD22 in the extracellular space with a dissociation constant less than 500 nM.

26. The endolysosomal targeting conjugate of claim 24 or claim 25 wherein the targeting component is configured to bind to cell surface CD22 with a lower dissociation constant at a near neutral pH than at an acidic endolysosomal pH.

27. The endolysosomal targeting conjugate of claim 26 wherein the near neutral pH is from about 6.8 to about 7.5 and the acidic endolysosomal pH is from about 5.0 to about 6.5.

28. The endolysosomal targeting conjugate of any of claims 24-27 wherein the targeting component comprises a Fab fragment or a scFv fragment of a CD22-specific antibody, wherein the Fab fragment or the scFv fragment comprises: a) a heavy chain variable domain of SEQ ID NO. 9, further comprising a mutation of Asn103 to histidine and / or b) a light chain variable domain of SEQ ID NO. 10, further comprising a mutation of Gln99 to histidine, Tyr101 to valine and / or Thr102 to valine.

29. A nucleic acid encoding the antibody or antibody fragment of any of claims 1 -23, or the targeting component of the endolysosomal targeting conjugate of any of claims 24-28.

30. An expression vector comprising the nucleic acid of claim 29.

31. A host cell comprising the expression vector of claim 30.

32. A pharmaceutical composition comprising the antibody or antibody fragment of any of claims 1-23, the endolysosomal targeting conjugate of any of claims 24-28, the nucleic acid of claim 29, the expression vector of claim 30, and / or the host cell of claim 31 ; and further comprising a pharmaceutically acceptable carrier.

33. The antibody or antibody fragment of any of claims 1-23, the endolysosomal targeting conjugate of any of claims 24-28, the nucleic acid of claim 29, the expression vector of claim 30, the host cell of claim 31 and / or the pharmaceutical composition of claim 32 for use in therapy.

34. The antibody or antibody fragment of any of claims 1-23, the endolysosomal targeting conjugate of any of claims 24-28, the nucleic acid of claim 29, the expression vector of claim 30, the host cell of claim 31 and / or the pharmaceutical composition of claim 32 for use in treating cancer.

35. The antibody or antibody fragment of any of claims 1-23, the endolysosomal targeting conjugate of any of claims 24-28, and / or the pharmaceutical composition of claim 32 for use according to claim 34, wherein the antibody or antibody fragment of any of claims 1-23 or the targeting component of the endolysosomal targeting conjugate of any of claims 24-28 is conjugated to a drug and / or a linker.

36. The antibody or antibody fragment of any of claims 1-23, the endolysosomal targeting conjugate of any of claims 24-28, the nucleic acid of claim 29, the expression vector of claim 30, the host cell of claim 31 and / or the pharmaceutical composition of claim 32 for use according to claim 34 or claim 35, wherein the cancer is a tumour expressing CD22.

37. The antibody or antibody fragment of any of claims 1-23, the endolysosomal targeting conjugate of any of claims 24-28, the nucleic acid of claim 29, the expression vector of claim 30, the host cell of claim 31 and / or the pharmaceutical composition of claim 32 for use according to claim 36, wherein the cancer is a B cell tumour expressing CD22.

38. The antibody or antibody fragment of any of claims 1-23, the endolysosomal targeting conjugate of any of claims 24-28, the nucleic acid of claim 29, the expression vector of claim 30, the host cell of claim 31 and / or the pharmaceutical composition of claim 32 for use according to any of claims 34-37, wherein the cancer is selected from the group consisting of: B-cell malignancies, B cell acute lymphoblastic leukaemia, non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B cell lymphoma.

39. A method of treating cancer in a subject, the method comprising administering to the subject the antibody or antibody fragment of any of claims 1-23, the endolysosomal targetingconjugate of any of claims 24-28, the nucleic acid of claim 29, the expression vector of claim 30, the host cell of claim 31 and / or the pharmaceutical composition of claim 32, in a therapeutically effective amount, to treat the cancer.

40. The method of claim 39, wherein the antibody or antibody fragment of any of claims 1- 23 or the targeting component of the endolysosomal targeting conjugate of any of claims 24- 28 is conjugated to a drug and / or a linker.

41. The method of claim 39 or claim 40, wherein the cancer is selected from the group consisting of: B-cell malignancies, B cell acute lymphoblastic leukaemia, non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B cell lymphoma.

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