HK1230923B - Conjugates comprising an anti-egfr1 antibody - Google Patents

Description

Conjugates containing anti-EGFR1 antibodies

Technical Field

The present invention relates to conjugates, pharmaceutical compositions and methods for treating or modulating the growth of tumor cells expressing EGFR1 in humans.

Background of the Invention

Boron neutron capture therapy (BNCT) is a non-invasive treatment for malignant tumors, such as primary brain tumors and head and neck cancers. In BNCT, patients are injected with a tumor-localizing drug loaded with non-radioactive boron-10 atoms. When the drug is irradiated with low-energy thermal neutrons, it emits biodestructive alpha particles and lithium-7 nuclei.

BNCT requires drugs, such as conjugates, that are high in boron-10 and can specifically localize to tumors. Such conjugates should be easy to produce, stable, soluble, and safe. However, providing such conjugates is complicated, for example, because some types of chemicals appear to be incompatible with compounds containing boron-10.

It was an object of the present invention to provide conjugates which have improved properties as compared to known conjugates and contain a high content of boron-10.

SUMMARY OF THE INVENTION

The conjugate of the present invention is characterized by what is presented in claim 1 .

The pharmaceutical composition of the present invention is characterized by what is presented in claim 18.

The conjugate or pharmaceutical composition of the invention for use as a medicament is characterized by what is presented in claim 19 .

The conjugate or pharmaceutical composition of the present invention for treating cancer is characterized by what is presented in claim 20 .

A method of treating or modulating the growth of tumor cells expressing EGFR1 in humans is characterized by what is presented in claim 22.

The prokaryotic host cell of the present invention is characterized by what is presented in claim 26 .

A method of treating or modulating the growth of tumor cells expressing EGFR1 in humans is characterized by what is presented in claim 56.

The polynucleotides of the present invention are characterized by what is presented in claims 57, 58, 59 and 60.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a further understanding of the present invention and constitute a part of this specification. The drawings illustrate embodiments of the present invention and together with the description help to explain the principles of the present invention. In the drawings:

Figure 1. Proton NMR spectrum of BSH-dextran. Boron-linked protons resonate between 0.8 and 2.0 ppm, and by comparing the integral of the boron-linked protons with the integral of the dextran protons, the boron loading of the BSH-dextran can be estimated. Unreacted allyl groups produce signals at 4.22, 5.29, 5.39, and 5.99 ppm. The sharp signal at 2.225 ppm is acetone (internal standard).

Figure 2. Gel filtration analysis of BSH-Dex-conjugate.

A. Anti-EGFR1-Fab-BSH(800B)-Dex. When analyzed using a Yarra SEC-3000 gel filtration column, the conjugate eluted in 7.8 ml. In comparison, anti-EGFR1-Fab eluted in 9.1 ml. B. Anti-EGFR1-Fab2-BSH(800B)-Dex. When analyzed using a Yarra SEC-3000 gel filtration column, the conjugate eluted in 6.9 ml. In comparison, anti-EGFR1-Fab2 eluted in 8.4 ml.

Figure 3. SDS-PAGE analysis of fluorescently labeled anti-EGFR1 Fab/F(ab') ₂ boron conjugates containing varying amounts of boron under non-reducing conditions (panel A) and reducing conditions (panel B). Anti-EGFR1-Fab-BSH-Dex conjugate: lane 1 (900B), lane 2 (700B), lane 4 (560B), lane 6 (360B). Anti-EGFR1-F(ab') _2- BSH-Dex conjugate: lane 3 (700B), lane 5 (560B), lane 7 (360B). Lane 8 is anti-EGFR1-Fab-Dex and lane 9 is a control containing a mixture of anti-EGFR1-F(ab') ₂ and an Fc fragment (Fab fragments migrate similarly on the gel). Gels were stained with Coomassie blue.

Figure 4. Cell surface binding and internalization of fluorescently labeled anti-EGFR1-F(ab') ₂ (panels A and C) and anti-EGFR1-F(ab') _2- BSH(900B)-Dex (panels B and D) by HSC-2 cells. Cells were incubated at +4°C (cell surface binding) and +37°C (cell surface binding and internalization). Analysis was performed by fluorescence microscopy.

Figure 5. Example of vector structure for signal peptide optimization. Identified T5 promoter, ribosome binding site (RBS), signal peptide, and anti-EGFR1 Fab heavy and light chain sequences.

Figure 6. Results of promoter optimization for Fab expression. 10 ml expression cultures were generated in liquid LB medium using W3110 pGF119 (A) or BL21(DE3) pGF121 (B). Induced cultures were incubated overnight at +20°C, and 1 ml samples were harvested and subjected to periplasmic extraction and subsequent Western blot analysis. 1) Background strain without expression vector; 2) W3110 pGF119 clone #1; 3) W3110 pGF119 clone #2; 4) W3110 pGF119 clone #3; C) 250 ng of control Fab.

Figure 7. Results of promoter optimization for Fab expression. 10 ml expression cultures were generated using W3110 pGF132 at three different post-induction temperatures: A) +20°C; B) +28°C; and C) +37°C. Different rhamnose concentrations were used for induction: 1) rha 0; 2) rha 0.25 mM; 3) rha 1 mM; 4) rha 4 mM; and 5) rha 8 mM. C = 100 ng control fab. Post-induction cultures were incubated at the indicated temperatures for 4 hours, and 1 ml samples were harvested and subjected to periplasmic extraction followed by Western blotting.

Figure 8. Comparison of bicistronic and dual promoter constructs. pGF119 and pGF121 are bicistronic vectors, while pGF120 and pGF131 are dual promoter vectors. 1) Uninduced control; 2) W3110 pGF119#1; 3) W3110 pGF119#2; 4) W3110 pGF120 uninduced; 5) W3110 pGF120#1; 6) W3110 pGF120#2; 7) Lemo21(De3) pGF131#1; 8) Lemo21(De3) pGF131#2; 9) Lemo21(De3) pGF121#1; 10) BL21(De3) pGF131#1; 11) BL21(De3) pGF131#2; C) 100 ng control fab.

Figure 9. Anti-EGFR1 Fab expression in E. coli Lemo21(De3) and BL21(DE3) containing the periplasmic chaperones SKP (pGF134) and SKP/FkpA (pGF135). Lemo21(De3) cultures were generated using built-in features of the strain that enable fine-tuning of rhamnose. Lane 1) Lemo21(De3) pGF131; 2) Lemo21(De3) pGF131 pGF134; 3) Lemo21(De3) pGF131 pGF135; 4) BL21(De3) pGF131; 5) BL21(De3) pGF131 pGF134; 6) BL21(De3) pGF131 pGF135; C) 100 ng of control Fab. At +28°C using 250uM rhamnose, Lemo21(De3)pGF131 pGF134 and –pGF135 (lanes 2 and 3) produced significantly more anti-EGFR1 Fab than Lemo21(De3)pGF131 (lane 1). At +20°C, BL21(De3)pGF131 pGF134 and –pGF135 (lanes 5 and 6) produced significantly more anti-EGFR1 Fab than BL21(De3)pGF131 (lane 4).

Figure 10. Western blot analysis of periplasmic expression of anti-EGFR1 Fab. Lane 1) Molecular weight marker; Lane 2) Anti-EGFR1 Fab control protein, 100 ng; Lane 3) Blank; Lane 4) Cell pellet sample before induction; Lane 5) Cell pellet sample 4 hours after induction; Lane 6) Cell pellet sample 16 hours after induction; Lanes 7-9) Blank; Lane 10) Culture supernatant sample before induction; Lane 11) Culture supernatant sample 4 hours after induction; Lane 12) Culture supernatant sample 16 hours after induction. All samples represent 10 μl of fermentor culture suspension. The concentration of anti-EGFR1 Fab in the periplasmic extract of fermentor-grown E. coli cells was estimated by comparing the band intensity in the cell pellet sample 16 hours after induction (Lane 6) with the band intensity of the control anti-EGFR1 Fab in Lane 2 (100 ng). Lane 6 was estimated to contain 300 ng of anti-EGR1 Fab: 300 ng/10 μl = 30 mg/L.

Figure 11. Chromatogram of HiTrap SP FF purified periplasmic extract. Fractions A5-A10 were pooled for further purification steps.

Figure 12. Chromatogram of a sample of protein L purification. Fractions A5-A7 were pooled.

Figure 13. SDS-PAGE analysis of purified anti-EGFR1 Fab. Samples were loaded in equal amounts (24 μL). Lane 1) Molecular weight marker; Lane 2) Anti-EGFR1 Fab produced by papain digestion; Lane 3) Fab sample produced by 10% E. coli; Lane 4) Fab sample produced by 40% E. coli. In lanes 3 and 4, the LC (upper) and HC (lower) bands are resolved. In lane 2, the Fab is glycosylated, and the LC and HC bands cannot be separated.

Figure 14. Binding of anti-EGFRl Fab (upper panel) or Fab BSH-dextran (lower panel) to EGFRl on microarray slides.

Detailed Description of the Invention

The present invention relates to a conjugate comprising an anti-EGFR1 antibody or an EGFR1 binding fragment thereof and at least one dextran derivative, wherein

The glucan derivative comprises at least one D-glucopyranosyl unit, wherein at least one carbon selected from carbon 2, 3 or 4 of the at least one D-glucopyranosyl unit is substituted with a substituent of the formula:

-O-(CH ₂ ) _n -SB ₁₂ H ₁₁ ^2- ,

wherein n is in the range of 3 to 10; and

The glucan derivative is bound to the anti-EGFR1 antibody or the EGFR1 binding fragment thereof via a bond formed by the reaction between at least one aldehyde group formed by oxidative cleavage of the D-glucopyranosyl unit of the glucan derivative and an amino group of the anti-EGFR1 antibody or the EGFR1 binding fragment thereof.

The conjugate is suitable for use in boron neutron capture therapy. "Boron neutron capture therapy" (BNCT) should be understood to refer to targeted radiotherapy in which non-radioactive boron-10 is irradiated with low-energy thermal neutrons to produce biodestructive alpha particles and lithium-7 nuclei. Non-radioactive boron-10 can be targeted by incorporating it into tumor-localizing drugs, such as tumor-localizing conjugates.

“EGFR1” herein should be understood to refer to human epidermal growth factor receptor 1 (EGFR1) having the sequence set forth in SEQ ID NO: 1.

"Anti-EGFR1 antibody" should be understood to refer to an antibody that specifically binds to EGFR1. The term "specific binding" refers to the ability of an antibody to distinguish EGFR1 from any other protein to such an extent that it only binds to or significantly binds to EGFR1 in a collection of multiple different proteins as potential binding partners. By way of example only, specific binding and/or kinetic measurements can be measured, for example, by using a surface plasmon resonance-based method on a Biacore device, by immunological methods such as ELISA, or by, for example, a protein microarray.

“EGFR1 binding fragment thereof” should be understood to refer to any fragment of an anti-EGFR1 antibody that is capable of specifically binding to EGFR1.

In one embodiment, the anti-EGFR1 antibody is cetuximab, imgatuzumab, matuzumab, nimotuzumab, necitumumab, panitumumab, or zalutumumab.

In one embodiment, the anti-EGFR1 antibody is cetuximab.

In one embodiment, cetuximab has the sequence set forth in SEQ ID NOs: 2 and 3.

In one embodiment, cetuximab comprises or consists of the sequence set forth in SEQ ID NOs: 2 and 3.

In one embodiment, the anti-EGFR1 antibody is nimotuzumab.

In one embodiment, nimotuzumab has the sequence set forth in SEQ ID NOs: 4 and 5.

In one embodiment, nimotuzumab comprises or consists of the sequence set forth in SEQ ID NOs: 4 and 5.

The anti-EGFR1 antibody can be, for example, scFv, a single domain antibody, Fv, a VHH antibody, a diabody, a tandem diabody, Fab, Fab', F(ab') ₂ , Db, dAb-Fc, taFv, scDb, dAb ₂ , DVD-Ig, Bs(scFv) ₄ -IgG, taFv-Fc, scFv-Fc-scFv, Db-Fc, scDb-Fc, scDb- _CH 3 or dAb-Fc-dAb. In addition, the anti-EGFR1 antibody or EGFR1 binding fragment thereof can be present in a monovalent monospecific, multivalent monospecific, bivalent monospecific, or multivalent multispecific format.

In one embodiment, the anti-EGFR1 antibody is a human antibody or a humanized antibody. In this case, the term "human antibody", as it is commonly used in the art, will be understood to mean an antibody having a variable region in which the framework region and the complementary determining region (CDR) are derived from human sequences. In this case, the term "humanized antibody", as it is commonly used in the art, will be understood to mean that the residues from the CDRs of human antibodies in the antibody are replaced by residues from the CDRs of non-human species (e.g., mouse, rat, or rabbit) with the desired specificity, affinity, and ability.

In one embodiment, the anti-EGFR1 antibody fragment comprises a Fab fragment of cetuximab. In one embodiment, the anti-EGFR1 Fab fragment has the sequence set forth in SEQ ID NOs: 6 and 3. In one embodiment, the anti-EGFR1 Fab fragment comprises or consists of the sequence set forth in SEQ ID NOs: 6 and 3.

In one embodiment, the anti-EGFR1 antibody comprises a F(ab') ₂ fragment of cetuximab. In one embodiment, the anti-EGFR1 F(ab') ₂ fragment has the sequence set forth in SEQ ID NOs: 7 and 3. In one embodiment, the anti-EGFR1 F(ab') ₂ fragment comprises or consists of the sequence set forth in SEQ ID NOs: 7 and 3.

"Glucan" is understood to refer to branched glucans comprising chains of varying lengths, wherein the linear chain consists of α-1,6 glycosidic bonds between D-glucopyranosyl units. Branches are joined via α-1,3 glycosidic bonds and, to a lesser extent, α-1,2 and/or α-1,4 glycosidic bonds. A portion of a linear glucan molecule is depicted in the following schematic diagram.

A "D-glucopyranosyl unit" is understood to refer to a single D-glucopyranosyl molecule. Glucans therefore contain a plurality of D-glucopyranosyl units. In glucans, each D-glucopyranosyl unit is linked to at least one other D-glucopyranosyl unit via an α-1,6 glycosidic bond, an α-1,3 glycosidic bond, or both.

Each D-glucopyranosyl unit of glucan contains 6 carbon atoms, which are numbered 1 to 6 in the following schematic. The schematic shows a single D-glucopyranosyl unit bonded to two other D-glucopyranosyl units (not shown) via an α-1,6 glycosidic bond.

Carbons 2, 3, and 4 may contain free hydroxyl groups. In a D-glucopyranosyl unit bonded to a second D-glucopyranosyl unit via an α-1,3 glycosidic bond, when carbon 3 of the D-glucopyranosyl unit is bonded to carbon 1 of the second D-glucopyranosyl unit via an ether bond, carbons 2 and 4 may be substituted with free hydroxyl groups. In a D-glucopyranosyl unit bonded to a second D-glucopyranosyl unit via an α-1,2 or α-1,4 glycosidic bond, when carbon 2 or 4 of the D-glucopyranosyl unit is bonded to carbon 1 of the second D-glucopyranosyl unit via an ether bond, carbons 3 and 4 or 2 and 3, respectively, may be substituted with free hydroxyl groups.

Sugar nomenclature is basically based on the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission (e.g., Carbohydrate Res. 1998, 312, 167; Carbohydrate Res. 1997, 297, 1; Eur. J. Biochem. 1998, 257, 293).

The term "glucan derivative" is understood to mean a glucan wherein at least one carbon selected from carbon 2, 3 or 4 of at least one D-glucopyranosyl unit is substituted by a substituent of the formula:

-O-(CH ₂ ) _n -SB ₁₂ H ₁₁ ^2- ,

wherein n is in the range of 3 to 10; and

The glucan derivative is bound to the anti-EGFR antibody or its EGFR1-binding fragment via a bond formed by the reaction between at least one aldehyde group formed by oxidative cleavage of the D-glucopyranosyl unit of the glucan derivative and an amino group of the anti-EGFR1 antibody or its EGFR1-binding fragment. The glucan derivative may also contain other modifications to the basic glucan structure, for example, as described below.

"BSH,""B ₁₂ H ₁₁ -SH," and "Na ₂ B ₁₂ H ₁₁ SH" should be understood to refer to sodium borocaptate, also known as sodium mercaptodecaborate and sulfhydrylborane. "B ₁₂ H ₁₁ ^2- " therefore refers to the borane portion of BSH.

One or more, ie, one, two or three carbons selected from carbons 2, 3 and 4 of at least one D-glucopyranosyl unit may be substituted with a substituent of the formula -O-(CH ₂ ) _n -SB ₁₂ H ₁₁ ^2- .

In one embodiment, n is 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, n is in the range of 3 to 4, or in the range of 3 to 5, or in the range of 3 to 6, or in the range of 3 to 7, or in the range of 3 to 8, or in the range of 3 to 9.

The D-glucopyranosyl units of glucans can be cleaved by oxidative cleavage of the bond between two adjacent carbon atoms substituted with a hydroxyl group. Oxidative cleavage cleaves vicinal diols, i.e., D-glucopyranosyl units in which two (free) hydroxyl groups occupy vicinal positions. D-glucopyranosyl units in which carbons 2, 3, and 4 contain free hydroxyl groups can therefore be oxidatively cleaved between carbons 2 and 3 or carbons 3 and 4. Thus, oxidative cleavage can be performed on a bond selected from the group consisting of the bond between carbons 2 and 3 and the bond between carbons 3 and 4. D-glucopyranosyl units of glucans can be cleaved by oxidative cleavage using oxidants such as sodium periodate, periodic acid, and lead (IV) acetate, or any other oxidant capable of oxidative cleavage of vicinal diols.

Oxidative cleavage of the D-glucopyranosyl unit forms two aldehyde groups, one at each end of the chain formed by oxidative cleavage. In the conjugate, the aldehyde group can, in principle, be a free aldehyde group. However, the presence of free aldehyde groups in the conjugate is generally undesirable. Therefore, the free aldehyde group can be capped or reacted with an amino group of an anti-EGFR1 antibody or its EGFR1 binding fragment or, for example, with a tracer molecule.

The glucan derivative is bound to the anti-EGFR antibody or the EGFR1-binding fragment thereof via a bond formed by the reaction between at least one aldehyde group formed by oxidative cleavage of the D-glucopyranosyl unit of the glucan derivative and an amino group of the anti-EGFR1 antibody or the EGFR1-binding fragment thereof.

The glucan derivative can also be bound to the anti-EGFR1 antibody or its EGFR1 binding fragment via a group formed by the reaction between at least one aldehyde group and an amino group of the anti-EGFR1 antibody or its EGFR1 binding fragment, wherein the at least one aldehyde group is formed by oxidative cleavage of the D-glucopyranosyl unit of the glucan derivative.

The aldehyde group formed by oxidative cleavage readily reacts with an amino group in solution, such as an aqueous solution. However, the resulting group or bond formed may vary and is not always easily predicted and/or characterized. The reaction between at least one aldehyde group formed by oxidative cleavage of the D-glucopyranosyl unit of the glucan derivative and an amino group of the anti-EGFR1 antibody or its EGFR1-binding fragment can, for example, result in the formation of a Schiff base. Thus, the group by which the glucan derivative binds to the anti-EGFR1 antibody or its EGFR1-binding fragment can, for example, be a Schiff base (imine) or a reduced Schiff base (secondary amine).

In one embodiment, the dextran derivative has a molecular mass in the range of about 3 to about 2000 kDa. In this case, the molecular mass of the dextran derivative should be understood to include the molecular mass of the dextran derivative containing dextran and its substituents, but not the molecular mass of the anti-EGFR1 antibody or EGFR1 binding fragment thereof. In one embodiment, the dextran derivative has a molecular mass in the range of 30 to about 300 kDa.

In one embodiment, the conjugate comprises from about 10 to about 300 or from about 20 to about ₁₅₀ ^substituents of the formula -O-( _CH2 ) _{n- SB12H112-} .

In one embodiment, the conjugate comprises about 300 boron atoms (300B), about 800 boron atoms (800B), about 900 boron atoms (900B), or about 1200 boron atoms. For example, "900B" refers to a conjugate that carries one mole of dextran per mole of protein, which carries about 900 moles of boron atoms in the BSH molecule.

The anti-EGFR1 antibody or EGFR1 binding fragment thereof generally contains at least one amino group, such as an N-terminal amine group and/or an amino group of a lysine residue.

In one embodiment, the amino group of the anti-EGFR1 antibody or EGFR1 binding fragment thereof is an amino group of a lysine residue of the anti-EGFR1 antibody or EGFR1 binding fragment thereof.

In one embodiment, the conjugate further comprises at least one tracer molecule bound to the dextran derivative or to the anti-EGFR1 antibody or EGFR1 binding fragment thereof.

"Tracer molecule" refers to a detectable molecule. Such a detectable molecule can be, for example, a radioisotope (e.g., ¹⁴ C), a compound containing a radioisotope, a radionuclide, a compound containing a radionuclide, a fluorescent marker molecule (e.g., FITC, TRITC, Alexa, and Cy dyes, etc.), a chelating agent, such as DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), or an MRI active molecule, such as gadolinium-DTPA (gadolinium-pentaacetic acid diethylenetriamine). Methods for achieving the binding of tracer molecules to dextran derivatives or to anti-EGFR1 antibodies or EGFR1 binding fragments thereof are well known in the art. The tracer molecule can allow the conjugate to be located after it has been administered to the patient and guided to specific cells; in this way, low-energy thermal neutron irradiation can be directed to the location of the guided conjugate.

In one embodiment, the tracer molecule is bound to the dextran derivative via a bond or group formed by the reaction between at least one aldehyde group formed by oxidative cleavage of the D-glucopyranosyl unit of the dextran derivative and a group of the tracer molecule. A suitable group of the tracer molecule may be, for example, an amino group.

One or more aldehyde groups formed by oxidative cleavage of the D-glucopyranosyl unit of the dextran derivative may not react with the amino group of the anti-EGFR1 antibody or the EGFR1 binding fragment thereof or with the tracer molecule.

In one embodiment, the glucan derivative comprises at least one blocked aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the glucan derivative.

The at least one aldehyde group may be blocked by a suitable group such as a reduced Schiff base.

The at least one aldehyde group may also be blocked by means of a group formed by the reaction between at least one aldehyde group and a hydrophilic blocking agent such as ethanolamine, lysine, glycine or Tris.

In one embodiment, the ethanolamine ^comprises14C .

The capping process can be stabilized using a reducing agent, such as NaCNBH _3. Capping groups such as reduced Schiff bases can thus be formed.

In one embodiment, the dextran derivative comprises at least one aldehyde group formed by oxidative cleavage of a D-glucopyranosyl unit of the dextran derivative, which aldehyde group does not react with an amino group of an anti-EGFR1 antibody or an EGFR1 binding fragment thereof or with a tracer molecule and is blocked.

In one embodiment, substantially all of the aldehyde groups formed by oxidative cleavage of one or more D-glucopyranosyl units of the glucan derivative are capped.

In one embodiment, the glucan derivative comprises a plurality of aldehyde groups formed by oxidative cleavage of D-glucopyranosyl units of the glucan derivative, wherein substantially all of the aldehyde groups formed by oxidative cleavage of one or more D-glucopyranosyl units of the glucan derivative are capped.

In one embodiment, at least one of carbons 2, 3 or 4 of at least one D-glucopyranosyl unit selected from the glucan derivative is substituted with a substituent of the formula:

-O-(CH ₂ ) _m CH＝CH ₂ ,

wherein m is in the range of 1 to 8. Although this embodiment is generally undesirable, it may appear as a by-product when the substituent has not yet reacted with BSH.

In one embodiment, the conjugate is obtainable by a process comprising the steps of:

a) alkenylating at least one hydroxyl group of a glucan to obtain an alkenylated glucan;

b) reacting borosulfate (BSH) with the alkenylated glucan obtainable from step a) to obtain BSH-glucan;

c) oxidative cleavage of at least one D-glucopyranosyl residue of the BSH-glucan, thereby forming an aldehyde group;

d) reacting the oxidatively cleaved BSH-dextran obtainable from step c) with an anti-EGFR1 antibody or an EGFR1 binding fragment thereof to obtain a conjugate.

The present invention also relates to a conjugate obtainable by a process comprising the steps of:

a) alkenylating at least one hydroxyl group of a glucan to obtain an alkenylated glucan;

b) reacting borosulfate (BSH) with the alkenylated glucan obtainable from step a) to obtain BSH-glucan;

c) oxidative cleavage of at least one D-glucopyranosyl residue of the BSH-glucan, thereby forming an aldehyde group;

d) reacting the oxidatively cleaved BSH-dextran obtainable from step c) with an anti-EGFR1 antibody or an EGFR1 binding fragment thereof to obtain a conjugate.

In one embodiment, the dextran has a molecular mass in the range of about 3 to about 2000 kDa, or about 10 to about 100 kDa, or about 5 to about 200 kDa, or about 10 to about 250 kDa. Dextran having a molecular mass in the said range should be understood to refer to dextran that has not undergone steps a) to d).

In this case, the term "alkenylation" or "alkenylation" should be understood to mean the transfer of an alkenyl group to a D-glucopyranosyl unit of a glucan to produce an alkenyl ether. That is, at least one hydroxyl group of a D-glucopyranosyl unit of a glucan is converted to an alkenyloxy group.

In step a), one or more hydroxyl groups bound to carbon 2, 3 or 4 of at least one D-glucopyranosyl unit of the glucan may react in an alkenylation reaction. One or more or multiple D-glucopyranosyl units of the glucan may be alkenylated.

In one embodiment, the glucan is alkenylated in step a) using an alkenylating agent, wherein the alkenylating agent has the structure of the following formula:

X-(CH ₂ ) _m CH＝CH ₂ ,

wherein m is in the range of 1 to 8, and X is Br, Cl or I.

In one embodiment, m is 1, 2, 3, 4, 5, 6, 7, or 8. In one embodiment, m is in the range of 1 to 2, or in the range of 1 to 3, or in the range of 1 to 4, or in the range of 1 to 5, or in the range of 1 to 6, or in the range of 1 to 7.

In one embodiment, the alkenylating agent is allyl bromide.

In one embodiment, at least one carbon selected from carbon 2, 3 or 4 of at least one D-glucopyranosyl unit of the alkenylated glucan obtainable from step a) is substituted with a substituent of the formula

-O-(CH ₂ ) _m CH＝CH ₂ ,

Here, m is in the range of 1 to 8.

In one embodiment, m is 1, 2, 3, 4, 5, 6, 7, or 8. In one embodiment, m is in the range of 1 to 2, or in the range of 1 to 3, or in the range of 1 to 4, or in the range of 1 to 5, or in the range of 1 to 6, or in the range of 1 to 7.

In step b), the sulfhydryl group of BSH can react with the alkenyl group of the alkenylated glucan to form BSH-glucan, thereby producing a thioether. One or more BSH molecules can react with the alkenylated glucan. Thus, the BSH-glucan obtainable from step b) can contain multiple BSH moieties (i.e., groups of the formula -SB ₁₂ H ₁₁ ^2- ). The sulfhydryl group of BSH can react with the alkenyl group of a single alkenylated D-glucopyranosyl unit containing more than one alkenyl group, or with the alkenyl groups of two or more alkenylated D-glucopyranosyl units.

Thus, the BSH-glucan obtainable from step b) may be a glucan derivative in which at least one carbon selected from carbon 2, 3 or 4 of at least one D-glucopyranosyl unit is substituted with a substituent of the formula:

-O-(CH ₂ ) _n -SB ₁₂ H ₁₁ ^2- ,

Wherein n is in the range of 3 to 10.

In one embodiment, the BSH-glucan obtainable from step b) comprises about 10 to about 100 or about 20 to ¹⁰⁰ substituents of formula -O-( _CH2 ) _n - _SB12H112- , or about 10 to about ₃₀₀ or about 20 to about 150 of the aforementioned substituents, wherein n is in the range of 3 to 10.

In one embodiment, BSH is reacted with the alkenylated glucan obtainable from step a) in step b) in the presence of a free radical initiator. The free radical initiator is capable of catalyzing the reaction between the sulfhydryl groups of BSH and the alkenyl groups of the alkenylated glucan.

In this context, "free radical initiator" is understood to refer to a chemical substance capable of generating free radical species and promoting free radical reactions under mild conditions. The term "free radical initiator" may also refer to UV (ultraviolet) light. Ultraviolet radiation can generate free radicals, for example in the presence of a suitable photoinitiator. Suitable free radical initiators include, but are not limited to, inorganic peroxides such as ammonium persulfate or potassium peroxodisulfate, organic peroxides, and ultraviolet light.

In one embodiment, BSH is reacted with the alkenylated glucan obtainable from step a) in step b) in the presence of a free radical initiator selected from ammonium persulfate, potassium peroxodisulfate and ultraviolet light.

In step b), the weight or molar ratio of BSH to the alkenylated glucan obtainable from step a) can be appropriately selected to obtain a conjugate in which the number of BSH moieties per glucan moiety (of the glucan derivative) (i.e., the number of substituents of the formula -O-(CH ₂ ) _n -SB ₁₂ H ₁₁ ^2- ) varies. The number of BSH moieties per glucan moiety of the BSH-dextran can be measured, for example, by nuclear magnetic resonance as described in Example 2 or by inductively coupled plasma mass spectrometry (ICP-MS) as described in Example 9.

In one embodiment, the ratio of BSH to alkenylated glucan present in step b) is in the range of 1:5 to 2:1 by weight, or in the range of 1:4 to 1:1 by weight, or in the range of 1:2 to 3:4 by weight. Generally, the higher the ratio of BSH to alkenylated glucan, the greater the number of BSH moieties per glucan moiety of the BSH-glucan.

The ratio of the free radical initiator (e.g., ammonium persulfate or potassium peroxodisulfate) in step b) may also be varied. In one embodiment, the ratio of free radical initiator to BSH and/or to dextran present in step b) is in the range of 1:5 to 2:1, or in the range of 1:4 to 1:1 by weight, or in the range of 1:2 to 3:4 by weight.

In one embodiment, the ratio of free radical initiator to alkenylated glucan present in step b) is in the range of 1 :5 to 2:1 , or in the range of 1 :4 to 1 :1 by weight, or in the range of 1 :2 to 3:4 by weight.

As described above, a bond selected from the group consisting of the bond between carbons 2 and 3 and the bond between carbons 3 and 4 can be oxidatively cleaved in step c). During the oxidative cleavage, the D-glucopyranosyl ring opens between the vicinal diols, leaving two aldehyde groups. The aldehyde groups of the oxidatively cleaved BSH-dextran obtained from step c) can be reacted with an anti-EGFR1 antibody or an EGFR1-binding fragment thereof to obtain a conjugate. The aldehyde groups can react with suitable groups, such as amino groups.

In principle, any oxidizing agent capable of oxidatively cleaving a D-glucopyranosyl unit between two adjacent carbon atoms substituted with a free hydroxyl group can be used to oxidatively cleave at least one D-glucopyranosyl residue of a BSH-glucan. The oxidizing agent can also be selected such that it oxidatively cleaves substantially specifically at least one D-glucopyranosyl residue of a BSH-glucan. Such an oxidizing agent may not oxidize other groups or moieties of the BSH-glucan.

In one embodiment, at least one D-glucopyranosyl residue of the BSH-glucan is oxidatively cleaved in step c) using an oxidizing agent selected from sodium periodate, periodic acid and lead (IV) acetate.

In one embodiment, at least one D-glucopyranosyl residue of the BSH-glucan is oxidatively cleaved in aqueous solution in step c).

In one embodiment, the method further comprises the step of reacting the oxidatively cleaved BSH-dextran obtainable from step c) or the conjugate obtainable from step d) with a tracer molecule.

In this case, the tracer molecule may be any tracer molecule described in this document.

The tracer molecule can react with at least one aldehyde group of the oxidatively cleaved BSH-dextran obtainable from step c).A suitable group of the tracer molecule that can react with at least one aldehyde group can be, for example, an amino group.

In one embodiment, the method further comprises the step e) of capping unreacted aldehyde groups of the oxidatively cleaved BSH-dextran obtainable from step c) or the conjugate obtainable from step d).

In one embodiment, unreacted aldehyde groups are capped using a hydrophilic capping agent such as ethanolamine, lysine, glycine, or Tris.

In one embodiment, the hydrophilic capping agent is selected from ethanolamine, lysine, glycine, and Tris.

In one embodiment, the ¹⁴ C-containing ethanolamine is the tracer molecule.

In one embodiment, one or more steps selected from steps a), b), c) and d) are carried out in an aqueous solution. A suitable aqueous solution may be, for example, an aqueous phosphate buffer having a pH of about 6 to 8.

In one embodiment, steps a) to d) are all performed in aqueous solution.

The anti-EGFR1 antibody or its EGFR1 binding fragment generally contains at least one amino group, such as an N-terminal amino group and/or an amino group of a lysine residue. In step d), the aldehyde group of the oxidatively cleaved BSH-dextran obtained from step c) can react with at least one amino group of the anti-EGFR1 antibody or its EGFR1 binding fragment.

In one embodiment, the amino group of the anti-EGFR1 antibody or EGFR1 binding fragment thereof is an amino group of a lysine residue of the anti-EGFR1 antibody or EGFR1 binding fragment thereof.

In one embodiment, the oxidatively cleaved BSH-dextran is reacted with the anti-EGFR1 antibody or its EGFR1 binding fragment by incubating the oxidatively cleaved BSH-dextran and the anti-EGFR1 antibody or its EGFR1 binding fragment in step d) at room temperature in an aqueous phosphate buffer solution having a pH of about 6 to 8.

The conjugate can be purified, for example, by gel filtration, eg, as described in Example 4.

The present invention also relates to producing anti-EGFR1 antibodies or EGFR1 binding fragments thereof in prokaryotic host cells. Compared with other polypeptide production systems, bacteria, especially Escherichia coli, provide many unique advantages. The raw materials used (i.e., bacterial cells) are cheap and easy to grow, thus reducing product costs. Prokaryotic hosts grow much faster than, for example, mammalian cells, thereby allowing for more rapid genetic manipulation analysis. The shorter generation time and ease of amplification also make bacterial fermentation a more attractive means of producing proteins in large quantities. The genome structure and biological activity of many bacterial species (including Escherichia coli) have been studied in depth, and a variety of suitable vectors are available, which makes it more convenient to express desirable antibodies. Antibody or antibody fragment expression in prokaryotic systems can be implemented on different scales. Shake flask culture (in the range of 2-5 liters) generally produces less than 5 mg/liter of product (e.g., antibody fragment), while 50-300 mg/liter scale can be obtained in fermentation systems.

Alternatively, prokaryotic host cells can produce aglycosylated anti-EGFR antibodies or EGFR1-binding fragments thereof.

In one embodiment, the prokaryotic host cell comprises one or more polynucleotides encoding an anti-EGFR1 antibody or an EGFR1 binding fragment thereof.

i) light chain variable region and

ii) Heavy chain variable region.

The term "one or more polynucleotides" may refer to two or more polynucleotides or polynucleotide molecules that may or may not be directly covalently linked or indirectly covalently linked via one or more sequences. For example, two or more polynucleotides may be contained in an expression cassette or vector. As an example, two or more polynucleotides may be fused directly or indirectly to encode a fusion protein comprising a light chain variable region and a heavy chain variable region. They may also be contained in two separate expression cassettes or vectors. The term "one or more polynucleotides" may also refer to a single continuous polynucleotide molecule that comprises one or more polynucleotides or polynucleotide fragments encoding the light chain variable region and the heavy chain variable region of an anti-EGFR1 antibody or its EGFR1 binding fragment.

In one embodiment, the host cell comprises a polynucleotide encoding an anti-EGFR1 antibody or EGFR1 binding fragment thereof according to one or more embodiments described herein. The host cell may comprise one or more polynucleotides that collectively encode an anti-EGFR1 antibody or EGFR1 binding fragment. The vector may be of any type, for example, a recombinant vector such as an expression vector.

Any of a variety of prokaryotic host cells can be used.

In one embodiment, the prokaryotic host cell is an E. coli cell.

In one embodiment, the one or more polynucleotides encoding the light chain variable region and the heavy chain variable region are codon-optimized for the host cell (eg, an E. coli cell).

In one embodiment, the host cell comprises a single contiguous polynucleotide encoding the light chain variable region and the heavy chain variable region of an anti-EGFR1 antibody or EGFR1 binding fragment thereof. Such a contiguous polynucleotide can be bicistronic or polycistronic.

In one embodiment, the prokaryotic host cell comprises a polynucleotide encoding a light chain variable region of an anti-EGFR1 antibody or EGFR1 binding fragment thereof and another polynucleotide encoding a heavy chain variable region of an anti-EGFR1 antibody or EGFR1 binding fragment thereof.

In one embodiment, the light chain variable region is preceded by a signal peptide. The polynucleotide thus encodes a signal peptide and a light chain variable region preceding the light chain variable region. The signal peptide may be immediately preceding the light chain variable region, or a sequence segment may be present between the signal peptide and the light chain variable region. The signal peptide may be selected from gIII, malE, phoA, ompA, pelB, stII, and stII. The signal peptide may also be selected from ompA, pelB, stII, and stII. These signal peptides may allow for particularly high productivity of the antibody or fragment in a prokaryotic host cell (e.g., E. coli).

In one embodiment, the heavy chain variable region is preceded by a signal peptide. The signal peptide can be selected from gIII, malE, phoA, ompA, pelB, stII, and stII. The signal peptide can also be selected from ompA, pelB, stII, and stII.

In one embodiment, the light chain variable region and the heavy chain variable region are preceded by a signal peptide.

In one embodiment, the signal peptide preceding the light chain variable region is different from the signal peptide preceding the heavy chain variable region.

In one embodiment, the signal peptides preceding the light chain variable region and the heavy chain variable region are independently selected from gIII, malE, phoA, ompA, pelB, stII and stII.

In one embodiment, the signal peptides preceding the light chain variable region and the heavy chain variable region are independently selected from mpA, pelB, stII and stII.

In one embodiment, the signal peptide preceding the light chain variable region is identical to the signal peptide preceding the heavy chain variable region, and wherein the signal peptide is selected from the group consisting of gIII, malE, phoA, ompA, pelB, stII and stII.

In one embodiment, the signal peptide preceding the light chain variable region is identical to the signal peptide preceding the heavy chain variable region, and wherein the signal peptide is selected from the group consisting of ompA, pelB, stII and stII.

In one embodiment, the light chain variable region is preceded by a pelB signal peptide and the heavy chain variable region is preceded by an ompA signal peptide.

In one embodiment, both the light chain variable region and the heavy chain variable region are preceded by a stII signal peptide.

In one embodiment, the polynucleotide comprises or consists of the sequence set forth in SEQ ID NO:8 and the sequence set forth in SEQ ID NO:9.

In one embodiment, the polynucleotide encoding the light chain variable region comprises the sequence depicted in SEQ ID NO:8, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8, and the sequence depicted in SEQ ID NO:9, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9, or consists of the sequence.

In one embodiment, the polynucleotide encoding the light chain variable region comprises or consists of the sequence set forth in SEQ ID NO:8 and the polynucleotide encoding the heavy chain variable region comprises or consists of the sequence set forth in SEQ ID NO:9.

In one embodiment, the polynucleotide encoding the light chain variable region comprises or consists of a sequence depicted in SEQ ID NO:8, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8, and the polynucleotide encoding the heavy chain variable region comprises or consists of a sequence depicted in SEQ ID NO:9, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9.

In one embodiment, the prokaryotic host cell comprises one or more polynucleotides encoding an anti-EGFR1 binding fragment of an antibody.

i) Light chain and

ii) Heavy chain.

In one embodiment, the one or more polynucleotides encode an anti-EGFR1 binding fragment Fab or scFv.

In one embodiment, the polynucleotide encoding the light chain comprises or consists of the sequence set forth in SEQ ID NO: 10, and the polynucleotide encoding the heavy chain sequence comprises or consists of the sequence set forth in SEQ ID NO: 11.

In one embodiment, the polynucleotide encoding the light chain comprises or consists of the sequence depicted in SEQ ID NO: 10, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, and the polynucleotide encoding the heavy chain sequence comprises or consists of the sequence depicted in SEQ ID NO: 11, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11.

In one embodiment, the one or more polynucleotides comprise or consist of the light chain sequence depicted in SEQ ID NO: 10 and the heavy chain sequence depicted in SEQ ID NO: 11.

In one embodiment, one or more polynucleotides comprise or consist of a light chain sequence depicted in SEQ ID NO: 10, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, and a heavy chain sequence depicted in SEQ ID NO: 11, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11.

In one embodiment, the host cell comprises a polynucleotide comprising or consisting of a sequence depicted in SEQ ID NO: 12, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.

In one embodiment, the host cell comprises a polynucleotide comprising or consisting of a sequence depicted in SEQ ID NO: 13, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13.

In one embodiment, the host cell comprises a chaperone protein and/or one or more polynucleotides encoding a chaperone protein. The chaperone protein can be a prokaryotic chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, FkpA and/or DsbG).

In one embodiment, the chaperone protein is overexpressed in the host cell.

In one embodiment, the chaperone protein is DsbA and/or DsbC.

In one embodiment, the chaperone protein is selected from the group consisting of DnaK, DnaJ, GrpE, Skp, FkpA, GroEL and GroES.

In one embodiment, the chaperone protein is Skp.

As used herein, the term "prokaryotic host cell" is intended to refer to a prokaryotic cell that has been or is capable of being genetically altered by the introduction of an exogenous polynucleotide (e.g., a recombinant plasmid or vector). It should be understood that such terms refer not only to the specific target cell, but also to the progeny of such a cell. Because certain modifications may appear in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "prokaryotic host cell" as used herein.

Prokaryotic host cells are transformed with the above-mentioned polynucleotides encoding anti-EGFR1 antibodies or EGFR1 binding fragments thereof (e.g., in expression vectors or cloning vectors) and cultured in conventional nutrient media, which, if appropriate, are adjusted to induce promoters, select transformants, or amplify genes encoding the desired antibody or antibody fragment sequences. Promoters suitable for use with prokaryotic hosts include PhoA promoters, β-lactamase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as tac or trc promoters. However, other promoters that are functional in bacteria (e.g., other known bacteria) are also suitable. Their nucleotide sequences have been published, allowing technicians to operably link them to cistrons encoding the light and heavy chains of interest (Siebenlist et al., (1980) Cell 20:269) using linkers or adapters providing any desired restriction sites.

In one embodiment, the one or more polynucleotides are driven by, ie, operably linked to, a promoter independently selected from the group consisting of T7, T5, and Rham.

In one embodiment, the one or more polynucleotides are driven by the promoter T7. Prokaryotic host cells used to produce anti-EGFR1 antibodies or EGFR1 binding fragments thereof can be cultured generally as described in the "Molecular Cloning" laboratory manual (Michael Green and Joseph Sambrook; 4th edition; Cold Spring Harbour Laboratory Press; 2012). Prokaryotic host cells suitable for expressing the antibodies of the invention include archaea and eubacteria, such as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacter, Pseudomonas species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, Gram-negative cells are used. In one embodiment, E. coli cells are used as hosts of the present invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, D.C.: American Society for Microbiology, 1987) pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 (U.S. Pat. No. 5,639,635) and strains 63C1 and 64B4 having the genotype W3110ΔfhuA(ΔtonA)ptr3lacIqlacL8ΔompTΔ(nmpc-fepE)degP41kanR. Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative and non-limiting. It may be necessary to consider the ability of the replicon to replicate in the bacterial cell and select an appropriate bacterium. For example, when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon, E. coli species can be appropriately used as the host. Generally, the host cell is likely to secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture.

In one embodiment, the host cell lacks one or more proteolytic enzymes.

In one embodiment, the proteolytic enzyme is selected from the group consisting of Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and Lon.

After transformation, the prokaryotic cells used to produce anti-EGFR1 antibodies or their EGFR1 binding fragments are cultured in a culture medium known in the art and suitable for cultivating the selected host cells. Examples of suitable culture medium include Luria broth (L), Terrific broth (TB) and synthetic minimal culture medium plus nutrient supplements such as yeast extract, soy hydrolyzate and other plant hydrolyzates. In some embodiments, the culture medium also contains a selection agent selected based on the expression vector construction process to selectively allow the prokaryotic cells containing the expression vector to grow. For example, ampicillin is added to the culture medium of cells expressing the ampicillin resistance gene. Any necessary supplements other than carbon source, nitrogen source and inorganic phosphate source can also be included in appropriate concentrations, the supplements being introduced alone or as a mixture with another supplement or culture medium such as a complex nitrogen source. Optionally, the culture medium can contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and dithiothreitol.

Prokaryotic host cells are cultured at a suitable temperature. For example, for E. coli growth, the preferred temperature range is from about 20°C to about 39°C. The pH of the culture medium can be anywhere from about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0. If an inducible promoter is used in the expression vector, expression of the anti-EGFR1 antibody or EGFR1 binding fragment protein is induced under conditions suitable for activating the promoter.

In one embodiment, anti-EGFR1 antibody or its EGFR1 binding fragment is secreted into the periplasm of prokaryotic host cells and recovered therefrom. Protein recovery generally includes destroying microorganisms by means such as osmotic shock, ultrasonic treatment or lysis. Once the cells are destroyed, cell debris or intact cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography or protein L column purification suitable for purification of Fab fragments. Alternatively, the protein can be transported to the culture medium and separated therein. The cell can be removed from the culture and filtered and the culture supernatant can be concentrated to further purify the protein produced. The expressed polypeptide can be further separated and identified using known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting assays.

In one aspect of the invention, anti-EGFR1 antibodies or EGFR1 binding fragments are produced in large quantities by fermentation methods. A variety of large-scale fed-batch fermentation methods can be used to produce recombinant proteins. Large-scale fermentation has a capacity of at least 500 liters. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (preferred carbon source/energy source). Small-scale fermentation generally refers to fermentation in a fermentor tank with a volume capacity no more than about 100 liters and can be from about 1 liter to about 100 liters.

In a fermentation method, induction of protein expression is generally initiated after the cells have been cultured to a desired density (e.g., OD ₅₅₀ of about 180-220) under suitable conditions, at which stage the cells are in an early stationary phase. As known in the art and described above, a variety of inducers can be used depending on the vector construct used. The cells can be cultured for a short time before induction. Typically, the cells are induced for about 12-50 hours, but longer or shorter induction times can be used.

To improve the production rate and quality of anti-EGFR1 antibodies or EGFR1 binding fragments, various fermentation conditions can be adjusted. For example, to improve the correct assembly and folding of secreted antibody polypeptides, additional vectors that overexpress chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and/or DsbG), Skp, or FkpA (peptidylprolyl cis, trans-isomerases with chaperone activity), can be used to co-transform host prokaryotic cells. Chaperone proteins have been shown to promote the correct folding and solubilization of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J. Biol. Chem. 274: 19601-19605; Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275: 17100-17105; Ramm and Pluckthun, (2000) J. Biol. Chem. 275: 17106-17113; Arie et al., (2001) Mol. Microbiol. 39: 199-210.

In one embodiment, chaperone proteins such as DnaK/DnaJ/GrpE, Skp, Skp/FkpA, GroEL/GroE are expressed in bacterial host cells such as E. coli.

In order to minimize the proteolysis of the expressed anti-EGFR1 antibody or its EGFR1 binding fragment (especially those that are sensitive to proteolysis), certain host strains lacking proteolytic enzymes can be used. For example, the host cell strain can be modified to achieve genetic mutations in genes encoding known bacterial proteases (e.g., Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof). Some E. coli strains lacking proteases are available and described in, for example, Joly et al., (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).

In one embodiment, an E. coli strain lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as a host cell in the expression system of the present invention.

Purification of anti-EGFR1 antibodies or EGFR1 binding fragments thereof can be achieved using methods recognized in the art. The following methods are examples of suitable purification methods: fractionation on an affinity chromatography column or an ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica gel or a cationic resin (e.g., DEAE), focusing chromatography, SDS-PAGE, ammonium sulfate precipitation, and gel filtration using, for example, Sephadex G-75.

In one embodiment, protein A immobilized on a solid phase is used for immunoaffinity purification of anti-EGFR1 antibodies.

In one embodiment, protein L immobilized on a solid phase is used for immunoaffinity purification of anti-EGFR1 antibody fragments of the present invention.

As a first step of purification, the preparation derived from the cell culture as described above is applied to a solid phase immobilized with Protein A or Protein L to allow specific binding of the anti-EGFR1 antibody to Protein A or the anti-EGFR1 antibody fragment (e.g., Fab fragment) to Protein L. The solid phase is then washed to remove impurities that are non-specifically bound to the solid phase. Finally, the antibody or antibody fragment is recovered from the solid phase by elution.

In one embodiment, the light chain variable region is preceded by a pelB signal peptide and the heavy chain variable region is preceded by an ompA signal peptide, the host cell comprises the chaperone protein Skp and/or a polynucleotide encoding the chaperone protein Skp; and the host cell lacks the proteolytic enzymes Lon and OmpT.

In one embodiment, the light chain variable region and the heavy chain variable region are preceded by a stII signal peptide, the host cell comprises the chaperone protein Skp and/or a polynucleotide encoding the chaperone protein Skp; and the host cell lacks the proteolytic enzymes Lon and OmpT.

Also disclosed are polynucleotides encoding anti-EGFR1 antibodies or EGFR1 binding fragments thereof.

i) light chain variable region and

ii) Heavy chain variable region.

In this case, the term "polynucleotide" can refer to one, two or more polynucleotides or polynucleotide molecules that can be directly covalently linked or can be indirectly covalently linked via one or more sequences. For example, two or more polynucleotides can be contained in an expression cassette or vector. As an example, two or more polynucleotides can be fused directly or indirectly to encode a fusion protein comprising a light chain variable region and a heavy chain variable region. They can also be contained in two independent expression cassettes or vectors. The term "polynucleotide" can also refer to a single continuous polynucleotide molecule that comprises one or more polynucleotides or polynucleotide fragments encoding a light chain variable region and a heavy chain variable region. The polynucleotide can be bicistronic or polycistronic.

In one embodiment, the one or more polynucleotides encoding the light chain variable region and the heavy chain variable region are codon-optimized for the host cell.The host cell can be a prokaryotic cell, such as an E. coli cell.

In one embodiment, the polynucleotide encoding the light chain variable region comprises or consists of a sequence depicted in SEQ ID NO: 8, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In one embodiment, the polynucleotide encoding the heavy chain variable region comprises or consists of a sequence depicted in SEQ ID NO: 9, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9.

In one embodiment, the polynucleotide encoding the light chain variable region comprises or consists of the sequence set forth in SEQ ID NO:8 and the polynucleotide encoding the heavy chain variable region comprises or consists of the sequence set forth in SEQ ID NO:9.

In one embodiment, the polynucleotide encoding the light chain variable region comprises or consists of a sequence depicted in SEQ ID NO:8, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8, and the polynucleotide encoding the heavy chain variable region comprises or consists of a sequence depicted in SEQ ID NO:9, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9.

In one embodiment, the polynucleotide encodes an anti-EGFR1 binding fragment of an antibody.

i) Light chain and

ii) Heavy chain.

In one embodiment, the anti-EGFR1 binding fragment encoded by the polynucleotide is a Fab or scFv.

In one embodiment, the polynucleotide comprises or consists of a light chain sequence depicted in SEQ ID NO: 10, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10.

In one embodiment, the polynucleotide comprises or consists of the heavy chain sequence depicted in SEQ ID NO: 11, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11.

In one embodiment, the polynucleotide comprises or consists of the light chain sequence depicted in SEQ ID NO: 10 and the heavy chain sequence depicted in SEQ ID NO: 11.

In one embodiment, the polynucleotide comprises, or consists of, a light chain sequence depicted in SEQ ID NO: 10, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, and a heavy chain sequence depicted in SEQ ID NO: 11, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11.

In one embodiment, the polynucleotide comprises or consists of the sequence depicted in SEQ ID NO: 12, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12.

In one embodiment, the polynucleotide comprises or consists of a sequence depicted in SEQ ID NO: 13, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13.

In one embodiment, the light chain variable region and the heavy chain variable region are preceded by a signal peptide. The polynucleotide thus encodes a signal peptide and a light chain variable region and a signal peptide and a heavy chain variable region. The two signal peptides can be selected independently of each other, or they can be the same signal peptide.

In one embodiment, the signal peptide preceding the light chain variable region is different from the signal peptide preceding the heavy chain variable region.

In one embodiment, the signal peptides preceding the light chain variable region and the heavy chain variable region are independently selected from gIII, malE, phoA, ompA, pelB, stII and stII.

In one embodiment, the signal peptides preceding the light chain variable region and the heavy chain variable region are independently selected from mpA, pelB, stII and stII.

In one embodiment, the signal peptide preceding the light chain variable region is identical to the signal peptide preceding the heavy chain variable region, and wherein the signal peptide is selected from the group consisting of gIII, malE, phoA, ompA, pelB, stII and stII.

In one embodiment, the signal peptide preceding the light chain variable region is identical to the signal peptide preceding the heavy chain variable region, and wherein the signal peptide is selected from the group consisting of ompA, pelB, stII and stII.

In one embodiment, the light chain variable region is preceded by a pelB signal peptide and the heavy chain variable region is preceded by an ompA signal peptide.

In one embodiment, both the light chain variable region and the heavy chain variable region are preceded by a stII signal peptide.

The polynucleotide can also be operably linked to a promoter, i.e., driven by it, or comprise the promoter. The promoter can allow for efficient expression of the polynucleotide. The promoter can also be an inducible promoter, thereby allowing for inducible expression of the polynucleotide.

In one embodiment, the polynucleotide is driven by, ie, is operably linked to, or comprises a promoter selected from the group consisting of T7, T5, and Rham.

In one embodiment, the polynucleotide is driven by or comprises a T7 promoter. In one embodiment, the prokaryotic host cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of an anti-EGFR1 antibody or an EGFR1 binding fragment of an anti-EGFR1 antibody. In one embodiment, the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of an anti-EGFR1 antibody or an EGFR1 binding fragment of an anti-EGFR1 antibody.

In one embodiment, the E. coli cells produce at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of anti-EGFR1 Fab.

In one embodiment, the E. coli cells produce at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of anti-EGFR1 scFv.

In one embodiment, the E. coli cell comprises the polynucleotide depicted in SEQ ID NO:8, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8, and the sequence depicted in SEQ ID NO:9, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9, or alternatively consists of the same, and the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of an anti-EGFR1 antibody or an EGFR1-binding fragment of an anti-EGFR1 antibody.

In one embodiment, the E. coli cell comprises the polynucleotide depicted in SEQ ID NO:8, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8, and the sequence depicted in SEQ ID NO:9, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:9, or alternatively consists of the same, and the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of anti-EGFR1 Fab or anti-EGFR1 scFv.

In one embodiment, the E. coli cell comprises or consists of the polynucleotide depicted in SEQ ID NO: 8 and the sequence depicted in SEQ ID NO: 9 and the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of anti-EGFR1 Fab or anti-EGFR1 scFv.

In one embodiment, the E. coli cell comprises the polynucleotide depicted in SEQ ID NO: 10, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, and the heavy chain sequence depicted in SEQ ID NO: 11, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11, or alternatively consists of the same, and the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of the anti-EGFR1 antibody or the EGFR1 binding fragment of the anti-EGFR1 antibody.

In one embodiment, the E. coli cell comprises the polynucleotide depicted in SEQ ID NO: 10, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10, and the heavy chain sequence depicted in SEQ ID NO: 11, or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 11, or alternatively consists of the same, and the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of anti-EGFR1 Fab.

In one embodiment, the E. coli cell comprises or consists of the polynucleotide depicted in SEQ ID NO: 12, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12, and the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of anti-EGFR1 scFv.

In one embodiment, the E. coli cell comprises or consists of the polynucleotide depicted in SEQ ID NO: 13, or a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 13, and the E. coli cell produces at least 20 mg/L, at least 30 mg/L, at least 50 mg/L, at least 100 mg/L, at least 200 mg/L, or at least 500 mg/L of anti-EGFR1 scFv.

The present invention also relates to a pharmaceutical composition comprising a conjugate according to one or more embodiments of the present invention.

The pharmaceutical compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutically acceptable carriers are well known in the art and may include, for example, phosphate-buffered saline solutions, water, oil/water emulsions, wetting agents, and liposomes. Compositions containing such carriers may be formulated by methods well known in the art. The pharmaceutical compositions may further comprise other components such as solvents, additives, preservatives, other pharmaceutical compositions to be administered concurrently, and the like.

In one embodiment, a pharmaceutical composition comprises an effective amount of a conjugate according to one or more embodiments of the invention.

In one embodiment, a pharmaceutical composition comprises a therapeutically effective amount of a conjugate according to one or more embodiments of the invention.

The term "therapeutically effective amount" or "effective amount" of the conjugate should be understood to refer to a dosage regimen that modulates cancer cell growth and/or treats the patient's disease when the cancer cells are bombarded with neutron radiation or exposed to BNCT. The therapeutically effective amount can be selected based on a variety of factors, including the patient's age, weight, sex, diet and medical condition, disease severity, and pharmacological considerations, such as the activity, potency, pharmacokinetic characteristics, and toxicological characteristics of the particular conjugate used. The therapeutically effective amount can also be determined by reference to standard medical texts (e.g., Physicians Desk Reference 2004). The patient can be male or female and can be an infant, child, or adult.

The terms "treatment procedure" or "treatment" are used in a conventional sense and mean accompanying, caring for and caring for a patient with the intention of preventing, reducing, diminishing or alleviating a disease or health disorder and improving living conditions impaired by such disease, for example, by cancer.

In one embodiment, the pharmaceutical composition comprises a composition for, for example, oral, parenteral, transdermal, intraluminal, intraarterial, intrathecal, intratumoral (i.t.) and/or intranasal administration or for direct injection into a tissue. Administration of the pharmaceutical composition can be achieved in different ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, intratumoral, topical or intradermal administration.

The present invention also relates to a conjugate according to one or more embodiments of the present invention or a pharmaceutical composition comprising a conjugate according to one or more embodiments of the present invention for use as a medicament.

The present invention also relates to a conjugate according to one or more embodiments of the present invention or a pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention, for use as a medicament for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is understood to be directed radiation therapy in which non-radioactive boron-10 is irradiated with low-energy thermal neutrons to produce alpha particles and lithium-7 nuclei. Non-radioactive boron-10 can be directed by incorporation into tumor-localizing drugs, such as tumor-localizing conjugates.

The present invention also relates to a conjugate according to one or more embodiments of the present invention or a pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention, for use in boron neutron capture therapy.

The present invention also relates to a conjugate according to one or more embodiments of the present invention or a pharmaceutical composition comprising a conjugate according to one or more embodiments of the present invention for use in treating cancer.

In one embodiment, the cancer is head and neck cancer.

In one embodiment, the cancer is selected from the group consisting of head and neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, small cell lung cancer, multidrug-resistant cancer, and testicular cancer.

The present invention also relates to a conjugate according to one or more embodiments of the present invention or a pharmaceutical composition comprising the conjugate according to one or more embodiments of the present invention, for use in boron neutron capture therapy for treating cancer.

The present invention also relates to the use of the conjugate or pharmaceutical composition according to one or more embodiments of the present invention for the manufacture of a medicament.

The present invention also relates to use of the conjugate or pharmaceutical composition according to one or more embodiments of the present invention for the manufacture of a medicament for boron neutron capture therapy.

The present invention also relates to the use of the conjugate or pharmaceutical composition according to one or more embodiments of the present invention for the manufacture of a medicament for treating cancer.

In one embodiment, the cancer is head and neck cancer.

In one embodiment, the cancer is selected from the group consisting of head and neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, small cell lung cancer, multidrug-resistant cancer, and testicular cancer.

The present invention also relates to the use of the conjugate or pharmaceutical composition according to one or more embodiments of the present invention for the manufacture of a medicament for treating cancer by boron neutron capture therapy.

In one embodiment, the medicament is for intratumoral treatment of head and neck cancer via boron neutron capture therapy.

In one embodiment, the medicament is for the intravenous treatment of head and neck cancer via boron neutron capture therapy.

In one embodiment, the medicament is used for the intratumoral and intravenous treatment of head and neck cancer via boron neutron capture therapy.

The present invention also relates to a method of treating or regulating the growth of tumor cells expressing EGFR1 in a human, wherein the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered to the human in an effective amount.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered to a human in an effective amount for boron neutron capture therapy.

In one embodiment, boron concentration in tumor cells is analyzed after administration of the conjugate or pharmaceutical composition.

In one embodiment, the boron concentration in the blood is analyzed after administration of the conjugate or pharmaceutical composition.

In one embodiment, boron concentration in muscle or other non-tumor tissue is analyzed following administration of the conjugate or pharmaceutical composition.

Boron concentration in tumor cells, in blood, or both can be analyzed or measured, for example, by inductively coupled plasma mass spectrometry (ICP-MS) or inductively coupled plasma atomic emission spectrometry (ICP-AES), such as described in Example 9. These methods measure the amount (in moles) or concentration of boron atoms in a sample.

Boron concentrations in tumor cells, in blood, or both can also be analyzed or measured indirectly, for example, by using an embodiment of the conjugate comprising a tracer molecule and analyzing or measuring the concentration of the tracer molecule. For example, if the tracer molecule is fluorescent or radioactive, the fluorescence or radioactivity of the tracer molecule can be measured or visualized.

In one embodiment, the boron concentration in tumor cells and in the blood is analyzed after administration of the conjugate or pharmaceutical composition, and the ratio of the boron concentration in the tumor cells to the boron concentration in the blood is greater than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 200:1, 210:1, 220:1, 230:1, 240:1, or 250:1.

In one embodiment, the boron concentration in tumor cells and in muscle, or other non-tumor tissue is analyzed after administration of the conjugate or pharmaceutical composition, and the ratio of the boron concentration in tumor cells to the boron concentration in muscle or other non-tumor tissue is greater than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 200:1, 210:1, 220:1, 230:1, 240:1, or 250:1.

In one embodiment, the ratio of boron concentration in tumor cells to boron concentration in blood, muscle, or other non-tumor tissue is the molar ratio of boron atoms in tumor cells to boron atoms in blood, muscle, or other non-tumor tissue.

The present invention also relates to a method for regulating the growth of a cell population expressing EGFR1 protein, wherein the method comprises the steps of:

The conjugate according to one or more embodiments of the present invention or the pharmaceutical composition according to one or more embodiments of the present invention is contacted with a cell population expressing EGFR1 protein.

In one embodiment, the cell population expressing EGFR1 protein is a cancer cell population or a tumor cell population.

In this context, the term "cancer cell population" should be understood to refer to one or more cancer cell populations.

The conjugate can be contacted with a cell population, such as a cancer cell, in vitro, in vivo, and/or ex vivo, including, for example, cancer cells of the plasma, lung, breast, colon, prostate, kidney, pancreas, brain, bone, ovary, testis, and lymphoid organs; more preferably, lung cancer, colon cancer, prostate cancer, plasma cell cancer, blood cancer, or colon cancer; "regulating the growth of a cancer cell population" includes inhibiting cell population proliferation to avoid the division of more cells; reducing the rate of cell division growth, such as compared to, for example, untreated cells; killing a cell population; and/or preventing a cell population (e.g., cancer cell) from metastasizing. The growth of a cell population can be regulated in vitro, in vivo, or ex vivo.

In one embodiment, the cancer is selected from the group consisting of head and neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, small cell lung cancer, multidrug-resistant cancer, and testicular cancer.

The present invention also relates to a method of treating and/or regulating the growth of tumor cells and/or preventing said tumor cells in a human, wherein the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered to the human in an effective amount.

In one embodiment, the effective amount is a therapeutically effective amount.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered to a human in an effective amount for boron neutron capture therapy.

In one embodiment, the tumor cell is selected from the group consisting of leukemia cells, lymphoma cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cell carcinoma cells, small cell lung cancer cells, head and neck cancer cells, multidrug resistant cancer cells and metastatic, testicular cancer cells, advanced stage, drug-resistant or hormone-resistant multidrug resistant cancer cells and various forms thereof.

The present invention also relates to a method of treating cancer in a human, wherein the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered to the human in an effective amount.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered to a human in an effective amount for boron neutron capture therapy.

In one embodiment, the effective amount is a therapeutically effective amount.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered intravenously to a human in a therapeutically effective amount for boron neutron capture therapy.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered intratumorally to a human in a therapeutically effective amount for boron neutron capture therapy.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered intratumorally and intravenously to a human in a therapeutically effective amount for boron neutron capture therapy.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments of the present invention is administered intratumorally in a therapeutically effective amount to a head and neck tumor in boron neutron capture therapy.

In one embodiment, the cancer is selected from the group consisting of head and neck cancer, leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, small cell lung cancer, multidrug-resistant cancer, and testicular cancer.

In one embodiment, the conjugate or pharmaceutical composition according to one or more embodiments comprises an anti-EGFR1 antibody or EGFR1 binding fragment thereof obtainable by a method comprising:

culturing the prokaryotic host cell according to one or more embodiments; and

Isolate and/or purify the anti-EGFR1 antibody or EGFR1 binding fragment thereof.

In one embodiment, the anti-EGFR1 antibody or EGFR1 binding fragment thereof of the conjugate or pharmaceutical composition according to one or more embodiments comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

The present invention also relates to a method for treating or regulating the growth of tumor cells expressing EGFR1 in humans, wherein the conjugate according to one or more embodiments or the pharmaceutical composition according to one or more embodiments is administered to humans in an effective amount. The embodiments of the present invention described above can be used in any combination with each other. Several embodiments can be combined to form another embodiment of the present invention. Products, uses or methods related to the present invention may comprise at least one embodiment of the present invention as described herein.

The conjugates according to one or more embodiments of the present invention have numerous advantageous properties.

The conjugates according to one or more embodiments of the present invention are relatively non-toxic in the absence of low energy neutron irradiation and have low antigenicity.

It contains a large number of boron-10 atoms per conjugate molecule. In addition, it shows relatively good water solubility.

The conjugates according to one or more embodiments of the present invention also exhibit good pharmacokinetics, having a moderate residence time in the blood, high uptake in cells to which they are targeted, and low uptake in cells to which they are not targeted.

Its production process is relatively simple and can be carried out in aqueous solution.

The conjugates according to one or more embodiments of the present invention are sufficiently stable to chemical or biochemical degradation during manufacturing or under physiological conditions, such as in serum, plasma, or tissue.

Example

Hereinafter, the present invention will be described in more detail. Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated, for example, in the accompanying drawings. The following description discloses some embodiments of the present invention in such detail that one skilled in the art can utilize the present invention based on the disclosure. Not all steps of the embodiments are discussed in detail, as many steps will be apparent to one skilled in the art based on this description.

Example 1. Allylation of dextran

200 mg of dextran 70 kD (Sigma) was dissolved in 2 ml of 0.6 M NaOH. 250 μl of allyl bromide (Sigma) was added and the reaction was allowed to proceed at 60° C. for 3 hours. The reaction mixture was then neutralized with 1 M acetic acid and the product was isolated by precipitation with 10 volumes of cold acetone (−20° C.). The precipitate was collected by centrifugation and washed twice with acetone. The allylated dextran (Scheme 1) was subjected to ¹ H-NMR analysis, which showed that the level of allylation was approximately 40%.

Scheme 1. Allylation of dextran using allyl bromide.

Example 2. Addition of BSH to Allyl Dextran

50 mg of allyl dextran 70 kD, 50 mg of ammonium persulfate and 50 mg of borosilicate sodium (BSH; Katchem Ltd, Czech Republic) prepared as described in Example 1 were dissolved in 0.5 ml of _H2O .

The reaction was allowed to proceed at 50°C for 2 hours. The reaction product, BSH-dextran, was isolated by ultrafiltration using a centrifugal filter (Amicon, 10K cutoff) (Scheme 2). ^1H -NMR analysis showed an average of 100 BSH units attached to the allyl dextran, corresponding to 1200 boron atoms per dextran chain (Figure 1). For example, by using a lower level of allylation in the dextran and employing minor modifications, BSH dextran with approximately 900 or 800 boron atoms per dextran chain was obtained.

Scheme 2. Addition of borocalcin sodium to allyl dextran in a persulfate-catalyzed reaction.

By varying the amounts of BSH and persulfate in the reactions described above, BSH-dextran with significantly lower BSH levels can be prepared: 1) In a reaction containing 20 mg allyl dextran, 15 mg ammonium persulfate, and 15 mg BSH, isolated BSH-dextran was found to contain approximately 700 boron atoms per glucan chain. 2) In a reaction containing 20 mg allyl dextran, 10 mg ammonium persulfate, and 10 mg BSH, isolated BSH-dextran was found to contain approximately 560 boron atoms per glucan chain. 3) In a reaction containing 20 mg allyl dextran, 5 mg ammonium persulfate, and 5 mg BSH, isolated BSH-dextran was found to contain approximately 360 boron atoms per glucan chain.

Example 3. Oxidation of BSH-dextran

50 mg of BSH-dextran, prepared as described in Example 2, was dissolved in 3 ml of 25 mM _NaIO4 in 0.1 M sodium acetate (pH 5.5). The reaction tube was covered with aluminum foil and incubated overnight at room temperature. The reaction product, oxidized BSH-dextran, was isolated by ultrafiltration using a centrifugal filter (Amicon, 10K cutoff) (Scheme 3).

Protocol 3. Oxidation of BSH-dextran by using sodium periodate.

Example 4. Conjugation of Oxidized BSH-Dextran to Anti-EGFR1 Fab/F(ab') ₂

2mg (40nmol) anti-EGFR1 Fab in 2ml phosphate buffered saline (PBS) is mixed with 5.1mg (60nmol) oxidized BSH-dextran (Example 3) in 1.6ml PBS. Allow the reaction to proceed overnight at room temperature. Add 400 μl 0.5M NaCNBH ₃ to the reaction to stabilize the aldehyde-lysine bond and incubate the reaction at room temperature for 2 hours. Add 800 μl 0.2M ethanolamine-HCl pH 8 and incubate the reaction at room temperature for 1 hour. Add 400 μl 0.5M NaCNBH ₃ to stabilize the ethanolamine blocking process and incubate the reaction at room temperature for 2 hours. Use PBS as washing eluent, remove low molecular weight reagents by Amicon centrifugal filter device (MWCO 30K) according to the manufacturer's instructions.

2 mg (40 nmol) anti-EGFR1 F(ab') ₂ in 2 ml phosphate buffered saline (PBS) was mixed with 2.56 mg (30 nmol) oxidized BSH-dextran (Example 3) in 1.6 ml PBS. The conjugate was stabilized, capped and purified by ultrafiltration as above.

Both conjugates were analyzed by purification (GE Healthcare) on a Yarra 3 μm SEC-3000 gel filtration column (300×7.8 mm; Phenomenex) using 10% acetonitrile (ACN)-50 mM Tris-HCl (pH 7.5) as elution buffer ( FIG. 2 ).

Example 5. Production of anti-EGFR1-Fab and anti-EGFR1-F(ab') ₂ and control-Fab fragments and control-F(ab') ₂ fragments

Fab fragments and F(ab') ₂ fragments were generated from commercial cetuximab (Erbitux, Roche) or cetuximab produced by CHO cells (Freedom CHO-S kit, Invitrogen). The Freedom CHO-S kit (Life Technologies) was used to develop a stable cell line producing cetuximab. This work was performed according to the manufacturer's instructions. Optimized nucleotide sequences encoding heavy and light chain sequences were purchased from GeneArt (Life Technologies) and cloned into the pCEP4 expression vector (Life Technologies), respectively. For stable expression, FreeStyle ^™ CHO-S cells were transfected with a linearized 1:1 light chain vector and heavy chain vector. Transfectants were selected with puromycin and methotrexate, and thereafter clones were isolated by limiting dilution cloning. The cloned cell lines were expanded and their productivity was evaluated.

Control-Fab fragment and control-F(ab') ₂ fragment were generated from commercial omalizumab (anti-IgE) (Xolair, Novartis).

Anti-EGFR1 Fab fragments were prepared by digesting the antibody with immobilized papain (Pierce) according to the manufacturer's instructions with minor modifications. The enzyme to substrate ratio used was 1:60 (% w/w) and the incubation time was 7 hours. According to the manufacturer's instructions, the Fab fragments were separated from undigested IgG and Fc fragments using immobilized protein A columns (Thermo Scientific).

Anti-EGFR1 F(ab') ₂ fragments were prepared by digesting the antibody with FragIT MaxiSpin (Genovis) according to the manufacturer's instructions or with Fabricator enzyme (Genovis) with minor modifications according to the manufacturer's instructions. Fabricator enzyme digestion was performed with 120 units of enzyme per mg of antibody in 50 mM sodium phosphate buffer pH 6.6 and the incubation time was 1 hour at +37°C. F(ab') ₂ fragments were purified using immobilized HiTrap protein L columns (GE Healthcare) according to the manufacturer's instructions. The reaction buffer was changed to PBS using an Amicon Ultra concentrator (Millipore) (10 kD cutoff).

The generated fragments were identified by SDS-PAGE and the protein concentration of each fragment was determined by measuring UV absorbance at 280 nm.

Example 6. SDS-PAGE analysis of boron conjugates

Boron conjugates of anti-EGFR1 Fab fragments and anti-EGFR1 F(ab') ₂ fragments were analyzed using SDS-PAGE to verify that conjugation was successful and that no unconjugated Fab or F(ab') ₂ fragments were present after conjugation. Figure 3 shows SDS-PAGE analysis of anti-EGFR1 Fab/F(ab') ₂ boron conjugates containing varying amounts of boron on gradient gels (Bio-Rad, 4-15%) under non-reducing conditions (panel A) and reducing conditions (panel B). The results in panel A indicate that conjugation was complete (or nearly complete) as no unconjugated Fab or F(ab') ₂ fragments were observed. BSH is a negatively charged molecule, and when conjugated to a protein, the conjugate migrates faster on the gel than would be expected based on its theoretical molecular weight. The example in Figure 3 (panel A) shows that a conjugate with a higher amount of boron migrates faster on a non-reducing gel than a conjugate with a lower amount of boron (e.g., compare lanes 1, 2, 4, and 6). The results of Figure 3 (panel A) also show that most conjugates separated into two bands on the non-reducing gel, suggesting that the sample contained a mixture of two different types of conjugates. SDS-PAGE analysis of the boron conjugates under reducing conditions (Figure 3, panel B) showed that all Fab conjugates with different boron amounts migrated similarly on the gel under reducing conditions (lanes 1, 2, 4, and 6). Similarly, reduced F(ab') ₂ conjugates with different boron amounts migrated similarly (lanes 3, 5, and 7). Overall, the reduced boron conjugates migrated faster on the gel than the unreduced conjugates.

Example 7. In vitro internalization assay of boron conjugates

AlexaFluor 488 labeling of boron conjugates

5 μg of AlexaFluor 488 carboxylic acid, succinimidyl ester label (Invitrogen) and 100 μg of boron conjugate (anti-EGFR1-Fab, anti-EGFR1-F(ab') ₂ , anti-EGFR1-mAb, control-Fab, control-F(ab') ₂ , control-mAb) or corresponding unconjugated compound were incubated for 15 minutes at room temperature in 100 μl PBS buffer containing 10 μl 1M NaHCO ₃ , pH 9. After incubation, excess label was removed by changing the buffer to PBS using an Amicon Ultra concentrator (Millipore) (10 kD cutoff). The protein concentration of each compound was determined by measuring the UV absorbance at 280 nm and the degree of labeling was calculated according to the manufacturer's instructions (Invitrogen).

Tritium labeling of boron conjugates

After removing the toluene solvent by evaporation, 100 μCi tritium-labeled N-succinimidyl propionate (PerkinElmer) and 100 μg anti-EGFR1-Fab-BSH (800B) -Dex, anti-EGFR1-F (ab ') ₂ -BSH (800B) -Dex, anti-EGFR1-mAb and control-mAb were incubated in 100 μl PBS (pH 8.8) buffer containing 20 μl 1M sodium borate. The reaction was allowed to proceed overnight at room temperature and subsequently the buffer was changed to PBS by adopting Amicon Ultra concentrator (10kD cutoff value) to remove excess label. The amount of radioactivity was measured with a scintillation counter in the presence of a scintillation counting fluid mixture (Ultima Gold, Perkin Elmer). The amount of tritium label in the compound was calculated as cpm/μg protein.

Cell culture

HSC-2 cells (human oral squamous cell carcinoma, JCRP Cellbank, Japan) and FaDu cells (human pharyngeal squamous cell carcinoma, ATCC) were cultured in T75 flasks in Eagle's minimal essential medium supplemented with 2% glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin. HEK (human embryonic kidney, ATCC) cells were cultured in T75 flasks in Dulbecco's modified Eagle's medium supplemented with 2% glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin.

Internalization assay visualized by fluorescence microscopy

HSC-2 cells (5x10 ⁴ ) were seeded on chamber slides and allowed to grow for 24 hours. Subsequently, cells were incubated for 3 hours at +37°C or at +4°C in 100 μl culture medium containing 10 μg/ml AlexaFluor 488-labeled BSH-conjugates. After incubation, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 20 minutes. Mounting medium (Prolong Gold fluorescence attenuation reagent containing DAPI) was added and cells were covered with microscope coverslips. Cell fluorescence microscopy (Zeiss Axio Scope A1; ProgRes C5, JENOPTIK AG) was used for photography.

The internalization of anti-EGFR1-F(ab') _2- BSH(900B)-Dex and unconjugated anti-EGFR1-F(ab') ₂ by the HSC-2 tumor cell line was analyzed by fluorescence microscopy (Figure 4). The experiments were performed at +4°C (compounds bound to the cell surface but unable to internalize) and at +37°C (cells were able to internalize surface-bound compounds). Both unconjugated anti-EGFR1-F(ab') ₂ and the boron conjugate bound to the cell surface at +4°C (panels A and B) and internalized at +37°C (panels C and D). In fact, the boron conjugate was more efficiently internalized than unconjugated anti-EGFR1-F(ab') ₂ . Internalization assays using anti-EGFR1-Fab-BSH(900B)-Dex and EGFR1-mAb-BSH(900B)-Dex and the corresponding unconjugated anti-EGFR1-Fab and anti-EGFR1-mAb gave results very similar to the data presented in Figure 4 (not shown). Boron conjugates with different boron amounts (anti-EGFR1-Fab-BSH-Dex and anti-EGFR1-F(ab') ₂ -BSH-Dex) were used to examine the effect of boron loading on internalization. The results showed that conjugates with more boron were more efficiently internalized by HSC-2 cells at +37°C than conjugates with lower boron loading (not shown). Control-F(ab') ₂ -BSH(900B)-Dex was only very weakly internalized (not shown).

Internalization assay (FACS)

HSC-2, FaDu and HEK cells (2x10 ⁵ ) were seeded on 24-well plates and allowed to grow for 24 hours. Subsequently, cells were incubated for 3 hours at +37°C in 300 μl culture medium containing a compound labeled with 5 μg/ml AlexaFluor 488. After incubation, cells were washed twice with PBS and detached from the culture medium by incubation with 100 μl trypsin-EDTA at +37°C for 10 minutes. Cells were neutralized by adding 300 μl culture medium and resuspended in PBS and analyzed using a flow cytometer (FACSLRS II). The mean fluorescence intensity of each sample was calculated using FACSDiva software. The data shown in Table 1-3 are described as "normalized mean fluorescence intensity," wherein fluorescence intensity is normalized to the degree of labeling of each compound.

FACS assay

The internalization of fluorescently labeled boron conjugates (900 boron atoms) and unconjugated Ab fragments was evaluated by FACS in the human HNC cancer cell line HSC-2. The results represent the internalization plus cell surface-bound compounds that occurred when the cells were incubated at +37°C (Table 1). Anti-EGFR1-Fab-BSH-Dex was more efficiently internalized than other boron conjugates or unconjugated anti-EGFR1-Fab. Other anti-EGFR1 boron conjugates (anti-EGFR1-F(ab') ₂ -BSH-Dex and anti-EGFR1-mAb-BSH-Dex) were equally well internalized as unconjugated anti-EGFR1-Fab and anti-EGFR1-F(ab') ₂ . The boron conjugates of control-F(ab') ₂ and control-mAb were very weakly internalized.

Table 1. Cell surface binding and internalization of fluorescently labeled boron conjugates and unconjugated compounds by HSC-2 cells. Analysis has been performed by FACS and the fluorescence intensity has been normalized to the degree of labeling of each compound.

Boron conjugates with different boron amounts (360-900 boron atoms) were synthesized from anti-EGFR1 F(ab') ₂ and anti-EGFR1-Fab to study the effect of boron loading on the internalization process. The examples show internalization assays of fluorescently labeled conjugates using the human HNC cancer cell line HSC-2 and the human control cell line HEK. The results from flow cytometry analysis represent the internalization plus cell surface-bound compounds that occurred when the cells were incubated at +37°C (Table 2). By flow cytometry analysis, the internalization of all boron conjugates of the anti-EGFR1Ab fragment was very similar. However, experiments using microscopy revealed that conjugates with more boron were more efficiently internalized than conjugates with low boron loading (not shown).

Table 2. Cell surface binding and internalization of fluorescently labeled boron conjugates with varying amounts of boron by HSC-2. Analysis has been performed by flow cytometry and the fluorescence intensity has been normalized to the degree of labeling of each compound.

Flow cytometry was used to evaluate the internalization of fluorescently labeled boron conjugates (1200 or 800 boron atoms) and unconjugated Ab fragments by human HNC cancer cell lines (HSC-2 and FaDu) and the control cell line HEK. The results represent the internalization plus cell surface-bound compounds that occurred when the cells were incubated at +37°C (Table 3). HSC-2 and FaDu cells showed the strongest internalization of anti-EGFR1-Fab-BSH(1200B)-Dex and unconjugated anti-EGFR1-Fab. FaDu cells internalized more weakly than HSC-2 cells, perhaps due to the lower amount of EGFR1 receptors on the cell surface. Control boron conjugates (control-Fab-BSH(800B)-Dex and control-F(ab') _2- BSH(800B)-Dex) and the corresponding unconjugated compounds were very weakly internalized. The control cell line HEK only very weakly internalized the boron conjugates and unconjugated compounds.

Table 3. Cell surface binding and internalization of fluorescently labeled boron conjugates (1200B or 800B) and unconjugated compounds by HSC-2 cells, FaDu and HEK cells. Analysis has been performed by flow cytometry and fluorescence intensity has been normalized to the degree of labeling of each compound.

Internalization assay of radiolabeled samples

HSC-2, FaDu and HEK cells (2x10 ⁵ ) were seeded on 24-well plates and allowed to grow for 24 hours. Subsequently, the cells were incubated at 37°C for 3 hours in 300 μl culture medium containing 5 μg/ml tritium-labeled compounds. After incubation, the culture medium was removed and the cells were washed 3 times with PBS and lysed by adding 300 μl 1M NaOH. The amount of radioactivity in the culture medium and cell lysates was measured using a scintillation counter in the presence of a scintillation counting fluid mixture (Ultima Gold). The amount of the internalized compound was calculated from the total amount of radioactivity in each well and normalized to 100,000 cells.

Boron conjugates (800 boron atoms) of anti-EGFR1 Fab and anti-EGFR1 F(ab') ₂ , as well as unconjugated anti-EGFR1 mAb, were labeled with tritium to lysine residues of protein components. Internalization assays of the radiolabeled compounds were performed using the human HNC cancer cell lines HSC-2 and FaDu, as well as the control HEK cell line. Results represent internalization plus cell-surface-bound compound when cells were incubated at +37°C. The results (Table 4) show that HSC-2 and FaDu cells internalized the boron conjugates of anti-EGFR1 Fab and anti-EGFR1 F(ab') ₂ as efficiently as unconjugated anti-EGFR1 mAb. Internalization by HSC-2 cells was 100-fold greater than that by FaDu cells, likely due to the higher amount of EGFR1 receptors on the cell surface in HSC-2 cells. The control HEK cell line showed only very weak internalization.

Table 4. Internalization of radiolabeled boron conjugates by HSC-2, FaDu and HEK cells. The amount of internalized compound has been calculated from the total amount of radioactivity per well and normalized to 100,000 cells. The results are the mean of three determinations +/- S.D.

Example 8. In vivo experiments of tritium-labeled conjugates

Preparation of mouse tissue and blood samples for liquid scintillation counting

Weighed mouse organs were dissolved in 1 ml of tissue dissolving agent (Solvable ^™ , Perkin Elmer) per 0.2 g of tissue. Samples were incubated at +60°C overnight. 150 μl of H ₂ O ₂ was subsequently added to every 300 μl of dissolved organs and samples and incubated at +60°C for 1 hour. The bones were first treated with 1 M HCl at +60°C overnight and then with Solvable and H ₂ O _2. The amount of radioactivity in the organs was measured using a scintillation counter in the presence of a scintillation counting fluid mixture (Ultima Gold ^™ , Perkin Elmer). The data are presented as the total injected dose % in grams of tissue. The results are the mean +/- SEM of three mice. Since each mouse had two tumors, the tumor results were the mean +/- SEM of six measurements.

Blood samples for the clearance study were collected in microcentrifuge tubes and the volume was measured after adding 100 μl of Solvable and incubating at +60°C overnight. 100 μl _{of H₂O₂} was then added and incubated at +60°C for 1 hour. The amount of radioactivity in the blood samples was measured using a scintillation counter in the presence of scintillation counting fluid cocktail (Ultima Gold, Perkin Elmer). Data are expressed as a percentage of the total injected dose. Results are the average of two mice.

Blood clearance of boron conjugates in non-tumor-free mice

Adult female mice of the same age (Harlan HSD: athymic nude mice Foxn1nu) were used. Radiolabeled ( ³ H) boron conjugates of anti-EGFR1-Fab and -F(ab') ₂ with 800B and 300B boron loadings were injected intravenously in 100 μl PBS via the tail vein. The injection dose was 30 μg = 1.3-2 x 10 ⁶ cpm/mouse and two mice were used per sample. Approximately 10 μl blood samples were collected at different time points before and after injection and the radioactivity was counted. At the end of the experiment (48 hours), the mice were sacrificed and organs were collected and radioactivity was counted to determine the tissue biodistribution of the conjugate.

Blood clearance studies in tumor-free mice were performed using ^3H -labeled boron conjugates of anti-EGFR1-Fab and -F(ab') ₂ with 800B and 300B boron loadings. Two different boron loadings were used to observe whether boron loading affected the clearance of the conjugate from the blood circulation. The results showed that the blood clearance of the boron conjugate was rapid and independent of boron loading (Table 5). The clearance process was comparable to that of the corresponding unconjugated F(ab') ₂ fragment and Fab fragment (not shown). Tissue distribution studies showed that the boron conjugate did not accumulate in any organ at 48 hours (not shown).

Table 5. Blood clearance of boron conjugates in non-tumor-free mice. Results are the average of two determinations. Times are times after administration (min) and values are % of the total injected dose.

Biodistribution of boron conjugates in mice with HSC-2 tumors

Adult female mice of the same age (Harlan HSD: athymic nude mice Foxn1nu) were used. Two and a half million to three million HSC-2 cells (JCRP Cellbank, Japan) in EME-medium and 50% matrigel were inoculated into the abdomen of the nude mice on both sides in 150 μl. Dosing was performed when at least one tumor in each mouse had grown to a size of at least 6 mm in diameter (6–10 mm) roughly corresponding to a tumor volume of 100-500 mm ^3. Radiolabeled ( ³ H) boron conjugates (800B) of anti-EGFR1-Fab/F(ab') ₂ and control-Fab/F(ab') ₂ were injected intravenously via the tail vein in 100 μl PBS. The dose injected was 50 μg = 1.3-2.6 x 10 ⁶ cpm/mouse and three mice/sample were used. Mice were sacrificed at different time points (24 hours, 48 hours, and 72 hours) and organs were collected and radioactivity was counted to determine tissue biodistribution of the conjugate.

The tissue distribution of the boron conjugates (Table 6) showed that the boron conjugates of anti-EGFR1-Fab and anti-EGFR1-F(ab') ₂ accumulated in the tumor, but not in any other organs, while the control boron conjugate did not significantly accumulate in the tumor. Tumor accumulation of the boron conjugates of anti-EGFR1-Fab and anti-EGFR1-F(ab') ₂ was highest at 24 hours and slowly decreased at later time points (48 hours and 72 hours).

Table 6. Biodistribution of boron conjugates in HSC-2 tumor-bearing mice. Results represent the mean +/- SEM of three determinations, with the exception of tumors where the mean +/- SEM is six determinations. Values are % of total injected dose/g organ.

Tumor to blood distribution of the boron conjugates in HSC-2 xenograft mice was calculated at different time points (24 hours, 48 hours, and 72 hours) (Table 7). For the anti-EGFR1-Fab conjugate, the tumor/blood ratio was 4-5, and for the anti-EGFR1-F(ab') ₂ conjugate, it was 2-6. The maximum ratio was reached earlier with anti-EGFR1-Fab-BSH-Dex (24 hours) than with anti-EGFR1-F(ab') ₂ -BSH-Dex (48 hours). The tumor/blood ratio of the control conjugate remained constant throughout the study (approximately 1-2).

Table 7. Tumor/blood distribution of boron conjugates in HSC-2 tumor-bearing mice. Results are based on the mean of three determinations for blood samples and the mean of six determinations (2 tumors per mouse) +/- S.D. for tumors.

Biodistribution of boron conjugates in mice with FaDu tumors

Adult female mice of the same age (Charles River Crl: athymic nude mice Foxn1nu) were used. Three million FaDu cells (ATCC) in EME-medium and 50% matrigel were inoculated into the abdomen of the nude mice on both sides in 150 μl. Dosing was performed when at least one tumor in each mouse had grown to at least 6 mm in diameter (6–10 mm, a size roughly corresponding to a tumor volume of 100-500 mm ³ ). Radiolabeled ( ³ H) boron conjugates (800B or 1200B) of anti-EGFR1-Fab/F(ab') ₂ and control-Fab/F(ab') ₂ were injected intravenously via the tail vein in 100 μl PBS. The dose injected was 50 μg = 2.3-2.7 x 10 ⁶ cpm/mouse, and three mice were used per sample. Mice were sacrificed at two different time points (24 hours and 48 hours) and organs were collected and radioactivity was counted to determine the tissue biodistribution of the conjugate.

Biodistribution studies in FaDu xenograft tumor-bearing mice were performed using boron conjugates of anti-EGFR1-F(ab') _2- BSH(800B)-Dex, anti-EGFR1-Fab (800B or 1200B)-BSH-Dex, and control-F(ab') ₂ and control-Fab (800B). The conjugates were radiolabeled (3H) to lysine residues of the protein. Radioactivity was counted in tissue samples (including tumor and blood) at two different time points (24 hours and 48 hours). Tissue distribution of the boron conjugates (Table 8) showed that the boron conjugates of anti-EGFR1-Fab and anti-EGFR1-F(ab') ₂ accumulated in the tumor but did not significantly accumulate in any other organs, while the control boron conjugate did not significantly accumulate in the tumor. Control-F(ab') _2- BSH(800B)-Dex was still present in the blood circulation and all organs at 24 hours, but was cleared from the circulation at 48 hours. Tumor accumulation of the boron conjugates of anti-EGFR1-Fab and anti-EGFR1-F(ab') ₂ was highest at 24 hours and decreased at 48 hours.

Table 8. Biodistribution of boron conjugates in FaDu tumor-bearing mice. Results represent mean +/- SEM of three determinations, except for tumors where the mean +/- SEM is six determinations. Values are % of total injected dose/g organ.

The tumor to blood distribution of the boron conjugates in FaDu xenograft mice was calculated at 24 hours and 48 hours (Table 9). The tumor/blood ratio for the anti-EGFR1-Fab conjugate and the anti-EGFR1-F(ab') ₂ conjugate with 1200 B of boron was approximately 7 at 24 hours, and this ratio dropped to 3-4 at 48 hours, indicating that the labeled protein was degraded and secreted out of the cell. The tumor/blood ratio of the anti-EGFR1-Fab conjugate with 800 B of boron was approximately 4-5 at the two time points. The tumor/blood ratio of the control conjugate remained at a constant level (approximately 1-2).

Table 9. Tumor/blood distribution of boron conjugates in FaDu tumor-bearing mice. Results are based on the mean of three determinations for blood samples and the mean of six determinations (2 tumors per mouse) +/- S.D. for tumors.

Boron conjugate 24 hours 48 hours anti-EGFR-Fab-BSH(800)-dex 4.4±2.2 5.5±1.5 anti-EGFR-Fab-BSH(1200)-dex 6.9±2.8 3.4±2.0 anti-EGFR-Fab2-BSH(1200)-dex 7.6±1.7 4.2±1.1 Control-Fab-BSH(800)-dex 1.8±0.5 1.5±0.2 Control-Fab2-BSH(800)-dex 1.7±0.4 1.2±0.4

Example 9. Quantification of Boron in BSH-Dextran by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) (mol boron/mol BSH-Dextran)

The boron loading of BSH-dextran was estimated from its proton-NMR spectrum ( FIG1 ), and ICP-MS was used to quantify the amount of boron in the sample. Based on NMR analysis, the BSH-dextran sample analyzed in the Examples was estimated to contain approximately 1200 boron atoms. Approximately 2.1 μg (0.0228 nmol) of BSH-dextran (average MW 92 kDa) was liquefied using microwave-assisted wet ashing and analyzed by ICP-MS essentially as described in Laakso et al., 2001, Clinical Chemistry 47, 1796–1803. Samples of varying dilutions were analyzed by ICP-MS, and background boron was subtracted from the samples. The results, representing the average of seven determinations, showed that the sample contained approximately 0.341 μg (31.5 nmol) boron atoms, or 1381 moles of boron atoms per mole of BSH-dextran.

Example 10. In vivo experiments and boron quantification

Female adult mice of the same age (Charles River Crl: athymic nude mice Foxn1nu) were used. Two million three hundred thousand HSC-2 or five million FaDu cells in EME-medium and 50% matrigel were inoculated into the right flank of the nude mice in 150 μl. When the tumor had grown to a size of at least 6 mm in diameter (6–10 mm, roughly corresponding to a tumor volume of 100-500 mm ³ ), administration was performed. Anti-EGFR-Fab-BSH(1200)-dex or anti-EGFR-F(ab') ₂ -BSH(1200)-dex (both unlabeled) conjugates were intravenously injected in 100 μl PBS via the tail vein. The dose injected was 50 μg or 250 μg/mouse and three mice were used per sample. The mice were sacrificed at 24 hours and 48 hours and organs were collected for boron determination.

Tissue samples (including blood) were digested in a sealed Teflon container in a microwave oven (Milestone, ETHOS 1200). The digestion temperature was 200°C and the duration of digestion was 50 minutes. The acid used in the digestion was _HNO₃ (6.0 ml, E. Merck, Suprapur). After cooling, the resulting solution was diluted to 25 ml with Milli-Q water. The digested samples were further diluted (1:10 or 1:50) with 1% _HNO₃ for ICP-MS analysis. Internal standard beryllium was added to the samples to achieve a final concentration of 10 ppb Be in the samples. Analytical standard solutions at concentrations of 1, 5, 10, _and 20 μg/L were diluted from Spectrascan single-element standard solutions (1000 μg/ml of boron in _H₂O as _H₃BO₃ ). Analytical control samples were prepared from multi-element standard solutions using SPEX (CLM-4). Analysis was performed using a high-resolution sector-field inductively coupled plasma mass spectrometer (HR-ICP-MS, Element2, ThermoScientific). Boron concentrations in the diluted samples were determined using the 10B and 11B peaks in low-resolution mode (R ≈ 300) and medium-resolution mode (R ≈ 4000). Between samples, the sample introduction system was flushed first with 5% HNO ₃ and then with 1% HNO ₃ to eliminate memory effects common to boron.

Initial boron analysis of two HSC-2 tumor mice at 24 hours showed boron tumor/muscle ratios of 5.3 and 6.3.

Muscle rather than blood was used as the control tissue because initial boron measurements from blood were inconclusive or exceeded the detection limit.

Example 11. In vivo experiments with ¹⁴ C-labeled anti-EGFR1 Fab BSH-dextran

Preparation of anti-EGFR1 Fab BSH-dextran

BSH-dextran was prepared as described in Examples 1 and 2, respectively. According to NMR analysis, BSH-dextran contained approximately 650 boron groups. Oxidation was performed as described in Example 3, but in two batches; one with 50 mg of BSH-dextran and the other with 100 mg of BSH-dextran.

Anti-EGFR1 Fab fragments were prepared by papain digestion as described in Example 5. Conjugation reactions were performed as in Example 4, but in four batches: 1) 29 mg oxidized BSH-dextran and 10.4 mg anti-EGFR1 Fab, 2) 16.5 mg oxidized BSH-dextran and 5.9 mg anti-EGFR1 Fab, 3) 50 mg oxidized BSH-dextran and 19.8 mg anti-EGFR1 Fab, and 4) 50 mg oxidized BSH-dextran and 19.7 mg anti-EGFR1 Fab, yielding a total of 55.8 mg anti-EGFR1 Fab. All were analyzed by SDS-PAGE as in Example 6, and each sample was labeled with Alexa Fluor 488-NHS. Internalization assays of Alexa Fluor 488-NHS-labeled molecules were performed using HSC-2 cells as described in Example 7.

Unlabeled Fab-BSH-dextran batches were pooled to yield 39 mg anti-EGFR1 Fab BSH-dextran. Prior to pooling of unlabeled ^and14C -labeled anti-EGFR1 Fab BSH-dextran and subsequent sterile filtration, the sample buffer was changed to 5% mannitol-0.1% Tween 80 in PBS.

Preparation of ¹⁴ C-labeled anti-EGFR1 Fab BSH-dextran

3 mg of Fab-BSH-dextran (before ethanolamine capping) was ¹⁴ C-labeled by incubation overnight with 66 μCi ¹⁴ C-ethanolamine (American Radiolabeled Chemicals Inc.) in PBS containing NaCNBH ₃ (as in Example 4), after which capping was completed with non-radioactive ethanolamine for 2 hours and the low molecular weight reagent was removed as described in Example 4. This reaction yielded ¹⁴ C-labeled anti-EGFR1 Fab BSH-dextran containing 9.21 μCi of radioactivity.

For animal studies, ¹⁴ C-labeled anti-EGFR1 Fab BSH dextran was mixed with unlabeled "cold" anti-EGFR1 Fab BSH dextran in the parts indicated in Table 10.

Table 10. Preparation of test materials.

In vivo experiments with ¹⁴ C-labeled anti-EGFR1 Fab BSH-dextran

Xenograft mice were generated as described in Example 8, except that HSC-2 cells were inoculated in the right flank and administration was performed when tumors had grown to a size of at least 8 mm in diameter (8–12 mm), roughly corresponding to a tumor volume of 200–800 mm ^3. Radiolabeled ( ¹⁴ C) anti-EGFR1-Fab boron conjugate was injected intravenously via the tail vein or by intratumoral injection (Group X) in 100 μl PBS containing 5% mannitol and 0.1% polysorbate (study groups are listed in Table 10). Three mice were used per sample. Approximately 400,000 cpm of conjugate was administered to each mouse (see above, Preparation of anti-EGFR1 Fab BSH dextran conjugate for animal studies; Table 10). Mice were sacrificed at 24 or 48 hours (Group IX) and organs were harvested and radioactivity counted to determine tissue biodistribution of the conjugate. Blood samples were also collected 30 minutes, 2 hours, and 8 hours after administration of the boron conjugate.

Tissues for ¹⁴ C quantification were prepared as described in Example 8. Blood samples for the clearance experiments were prepared as in Example 8, except that 200 μl of Solvable and 90 μl of H ₂ O ₂ were used. Results are the average of three mice.

Table 11 shows the tumor-to-blood ratios of mice administered with ¹⁴ C-labeled anti-EGFR1 Fab dextran conjugate.

Table 11: Tumor/blood ratios of ¹⁴ C boron conjugates in mice bearing HSC-2 tumors. Values for G IX are tumor/brain ratios because 0% radioactivity was measured in the blood (all blood samples were negative after background subtraction). Group I: 250 μg; Group II: 500 μg; Group III: 1000 μg; Group IV: 1500 μg; Group V: 2000 μg; Group X: 250 μg; Group VIII: 250 μg + 250 μg 2 hours later; and Group IX: 250 μg + 250 μg 24 hours later. All groups were administered intravenously, with the exception of Group X, which was administered intratumorally. Organs were harvested at 24 hours, with the exception of Group IX, which was harvested at 48 hours. Group VIII tumor/blood ratio from one mouse (due to the presence of one blood cpm value for this group).

G I G II G III G IV G V G X G VIII G IX 11.2 12.8 9.7 23.8 28.8 4394.3 9.3 6.2

Table 12. Blood clearance ^of14C boron conjugates in the three groups. The left column shows the time after administration (min/hour) and the values are % of the total injected dose/g blood.

Example 12. In vivo experiments with anti-EGFR1 Fab BSH-dextran by direct boron quantification

Preparation of anti-EGFR1 Fab BSH-dextran

Anti-EGFR1 Fab BSH-dextran was prepared as described in Examples 1 and 2, respectively. Oxidation was performed as described in Example 3, but in two batches; one with 80 mg of BSH-dextran and the other with 96 mg of BSH-dextran. According to NMR analysis, the BSH-dextran samples contained approximately 880 and 500 boron atoms, respectively.

Anti-EGFR1 Fab fragments were prepared by papain digestion as described in Example 5. Conjugation reactions were performed as in Example 4, but in four batches: two with 15.7 mg anti-EGFR1 Fab and 40 mg oxidized BSH-dextran and two with 18.8 mg anti-EGFR1 Fab and 48 mg oxidized BSH-dextran.

All boron conjugates were analyzed in SDS-PAGE and labeled with Alexa 488-NHS as in Example 6. HSC-2 cell internalization assays were performed as described in Example 7 using Alexa Fluor labeled molecules.

Prior to mouse study sample preparation and sterile filtration, the sample buffer was changed to 5% mannitol-0.1% Tween 80 in PBS.

In vivo experiments with anti-EGFR Fab BSH-dextran

Xenograft mice were generated as in Example 11. Anti-EGFR Fab BSH-dextran was administered intravenously in 100 μl of mannitol/Tween/PBS solution or intratumorally (i.t.) in 40 μl of mannitol/Tween/PBS solution. In intratumoral administration, the needle was passed into the tumor through a single injection site and moved using a fine needle aspiration technique to distribute the test substance throughout the tumor. A total of three or four passes were used, depending on tumor size and shape.

Organs were harvested at 24 hours and blood samples were collected at 30 minutes, 2 hours and 8 hours (Study Groups II and V).

Boron quantification

Tissue was prepared for direct boron quantification by ICP-MS as described above. Three control samples containing approximately 150 mg of NIS reference standard 1573 tomato leaves were also digested. The digested samples were diluted to 1:10 or 1:100.

Table 13 shows boron in selected organs and Table 14 shows tumor to blood ratios. Intratumoral administration showed significantly higher tumor boron concentrations compared to intravenous administration.

Table 13. Biodistribution of anti-EGFR1 Fab BSH-dextran conjugate in HSC-2 tumor-bearing mice based on boron quantification. Results represent the mean +/- SEM of four determinations. The study groups were: Group I: buffer alone (mannitol/Tween/PBS), intravenous; Group II: 2 mg, intravenous; Group III: 2 mg + dextran, intravenous; Group IV: 250 μg, intratumoral; Group V: 2 mg, intratumoral. Values are μg boron per g organ. Students' t-tests (using Statistica 12 software [StatSoft]) were performed on tumor boron values for Groups II and III and for Groups IV and V. Groups IV and V showed significant differences in boron amounts (p-value = 0.009).

Group II Group III Group IV Group V Group I blood 0.56±0.18 0.87±0.14 0.1±0.05 0.22±0.01 0.32±0.21 liver 18.3±1.25 17.54±1.15 1.02±0.33 6.97±0.86 0.27±0.09 kidney 6.57±0.57 6.44±0.41 0.87±0.27 3.78±0.24 0.76±0.31 muscle 1.87±0.34 1.51±0.9 0.56±0.25 0.7±0.31 0.87±0.51 skin 2.11±0.16 1.46±0.14 0.43±0.13 1.27±0.85 0.17±0.09 tumor 2.19±1.01 9.54±8.59 9.22±2.3 53.09±11.45 0.62±0.53 spleen 4.95±0.9 5.91±0.88 2.01±0.5 1.88±0.59 1.34±0.53

Table 14. Tumor to blood ratios +/- SEM.

Group II Group III Group IV Group V Group I 10.8±7.1 13.6±12.5 131.3±40.7 240.8±49.4 4.6±3.6

Example 13. Production of anti-EGFR1 Fab in E. coli

Optimization of signal peptide for periplasmic secretion of anti-EGFR1 Fab

The expression strategy of anti-EGFR1 Fab was to target the periplasm, where stable disulfide bonds can be formed.

The commercial vector kit pDD441-SSKT (T5 promoter, kanamycin selection) was used for signal peptide optimization. The following signal peptides were used: i) MalE (maltose binding protein), ii) pelB (pectate lyase), iii) ompA (outer membrane protein A), iv) phoA (bacterial alkaline phosphatase) and v) gIII (PRV envelope glycoprotein). Vectors pGF115–pGF119 were constructed by routinely using synthetic DNA sequences, PCR amplification with high-fidelity polymerases and seamless Gibson assembly. In addition, vector pGF150 containing the signal peptide stII (heat-stable enterotoxin II) for the heavy and light chains was constructed according to Carter et al. 1992: High level E. coli expression and production of bivalent humanized antibody fragments, Biotechnology (NY), 10(2):163-7. Vector pGF150 is bicistronic and has a T7 promoter for expression. The anti-EGFR1 Fab expression cassette is bicistronic, with an internal ribosome binding site between the heavy and light chains. Figure 5 illustrates a general expression vector structure used for signal peptide optimization. Table 15 lists the signal peptide combinations used in vectors pGF115–pGF119.

Table 15. Signal peptide combinations in vectors pGF115–pGF119.

The vector pGF115–pGF119 was transformed into electrocompetent E. coli W3110 (ATCC Microbiology Collection) cells using a Biorad GenePulser using program Ec2 pulse according to the manufacturer's instructions. The transformants were plated onto LB+agar+kanamycin 25 mg/L and grown overnight at +37°C.

Individual colonies were screened for expression according to standard protocols. On day 1, the overnight preculture was inoculated into 5 mL of liquid LB supplemented with kanamycin at a final concentration of 20 mg/L and incubated at +37°C with shaking at 220 rpm. On day 2, 200 μL of the overnight preculture was inoculated into 10 mL of liquid LB supplemented with 10 mg/L kanamycin. Cultivation was continued at +37°C with shaking at 220 rpm until the _OD600 reached 0.6–0.9. Fab production was induced with IPTG at a final concentration of 500 μM. Cultivation was continued at +20°C with shaking at 220 rpm overnight. 1 mL samples were collected at 4 hours and overnight post-induction. Cells were harvested by centrifugation at 8000 x G for 10 minutes, the supernatant discarded, and the pellet resuspended in 100 μL of 10x TE pH 7.5 (100 mM Tris-HCl, 10 mM EDTA). The samples were vortexed vigorously for 1 hour at room temperature, pelleted at 16,000 x G for 10 minutes and the supernatant collected as the periplasmic extract into a new microcentrifuge tube.

Periplasmic extracts were further analyzed by Western blotting. 100 μl of extract was mixed with 20 μl of reducing or non-reducing loading buffer. 20 μl of the mixture was loaded onto a 4-20% Precise Tris-glycine SDS-Page gel (Thermo Scientific). The gel was run at 200V for approximately 45 minutes in 1x Laemmli running buffer and blotted onto a nitrocellulose membrane at 350 mA for approximately 5 minutes in Tris-glycine blotting buffer. The BioRad Mini-protean system was used for SDS-Page and blotting. The blotted membrane was blocked with 1% BSA in PBS. Detection was performed using anti-human IgG (Fab specific) together with a peroxidase conjugate (Sigma Aldrich; catalog number A0293) and Luminata Forte Western Blot HRP substrate (Millipore; catalog number WBLUF0500). Chemiluminescent reactions were detected using a Fujifilm luminescent image analyzer LAS4000.

Based on Western blot analysis of several expression cultures, vectors pGF119 and pGF115 appeared to be superior to the others. However, in these preliminary experiments, the amount of Fab produced in the periplasm remained at a level of 0.3–0.8 mg/L. The combination used in vector pGF119 (ompA signal peptide for HC and pelB signal peptide for LC) was selected for continued work.

The vector pGF150 containing the T7 promoter and the signal sequence stII for targeting the heavy and light chains of the anti-EGFR1 Fab to the periplasm was transformed into the strain BL21 (DE3). Compared with other vectors, it appeared to be at least as good as the vector pGF119 using pelB for the light chain and ompA for the heavy chain.

Optimizing promoters for Fab expression

Three different promoters were used in the initial screening: IPTG-inducible T5, IPTG-inducible T7, and rhamnose-inducible Rham. Promoter sequences were derived from the commercial vectors pET-15b, pD441, and pD881. The signal peptides ompA were used for the HC and pelB for the LC. The expression cassettes were constructed in a bicistronic fashion, with the internal ribosome binding site taaGGATCCGAATTCAAGGAGATAAAAAatg (SEQ ID NO: 22) located between the heavy and light chains in each vector. Vector codes and promoters are shown in Table 16.

Table 16: Promoter system optimization for Fab expression; vector codes and promoters used.

carrier promoter pGF119 T5 pGF121 T7 pGF132 Rham

pGF119 and pGF132 were electroporated into E. coli strain W3110 as described above. The T7 promoter vector pGF121 was transformed into chemically competent E. coli BL21 (DE3) cells (New England Biolabs) according to the heat shock protocol provided by the supplier. Expression culture, sample preparation, and periplasmic extract analysis were performed as described above. First, comparisons were made between strain W3110 pGF119 and BL21 (De3) pGF121. Periplasmic extracts were generated in parallel using 10X TE buffer and 0.05% deoxycholate buffer.

As exemplified in Figure 6, the T7 promoter was slightly better than the T5 promoter, but the difference was not very significant. Repeated experiments using strains W3110 pGF119 and BL21 (De3) pGF121 revealed that expression cultures with BL21 (DE3) pGF121 were more stable and reproducible than those with W3110 pGF119. Faster growth rates and higher cell densities were achieved with BL21 (De3) pGF121 than with W3110 pGF119 (data not shown).

The second step in the promoter screening is to analyze the preliminary expression level from the small-scale culture of W3110 pGF132 (rhamnose inducible promoter). One of the advantages of the rhamnose inducible promoter is that the expression level can be finely adjusted by varying the rhamnose concentration. For some target proteins, lower expression levels actually result in higher total titers because the target protein correctly folds and assembles and the cell density of the production strain is higher. Therefore, induce with the rhamnose (0, 0.25mM, 1mM, 4mM and 8mM) of increasing concentration in parallel 10ml liquid LB cultures. Use three different post-induction temperatures: +20 ℃, +28 ℃ and +37 ℃. At the time point of 4 hours after induction, gather in the crops 1ml sample. Sampling, periplasmic extract and analysis are carried out as described in Example 1.

As shown in Figure 7, expression levels using the rhamnose-inducible promoter were still lower than those achieved with BL21(De3)pGF121 (T7 promoter). Regulation of the promoter with increasing concentrations of rhamnose was most efficient at +20°C. However, the highest titers were achieved with the rhamnose system at +28°C.

Based on the repeated experiments described above, the BL21(DE3) and T7 promoter system was chosen as the basic platform for the production of anti-EGFR1 Fab in E. coli.

Codon optimization of anti-EGFR1 Fab for expression in E. coli cells

Three HC/LC sequences with different codon optimization patterns for E. coli and one HC/LC sequence originally optimized for CHO cells were tested. Vectors were constructed as described for pGF119, a bicistronic approach, and T5 promoter-driven expression. The expression host was E. coli W3110. Small-scale cultivation, sampling, and periplasmic extract analysis were performed as described above. The sequence in vector pGF119 was selected as the baseline level. The codon optimization pattern had a significant effect on expression levels (Table 17). The E. coli version 2 sequence (pGF128) and the CHO cell-optimized sequence (pGF126) did not work in the W3110 host strain, with only measured Fabs detected from expression cultures by Western blotting. The expression levels achieved with E. coli version 3 (pGF129) were significantly better, but still similar to baseline levels. Because most vectors had been produced using E. coli version 1 (pGF119) and because no improvement was achieved by changing the codon optimization pattern compared to the baseline, the E. coli version 1 sequence from vector pGF119 (SEQ ID NO: 10 and SEQ ID NO: 11) was chosen.

Table 17. Anti-EGFR1 Fab coding sequences with different codon optimization patterns tested. Vector encoding and results.

Compare bicistronic vector structures to dual promoter vector structures.

In the bicistronic vector structure, the spacer sequence (comprising the ribosome bind site) between the heavy chain and the light chain is relatively short, with only 25 nucleotides in pGF119. In order to expand this spacing between the heavy chain and the light chain, vectors pGF120 and pGF131 were constructed, in which the two chains were expressed under the control of independent T5 or T7 promoters. By utilizing the existing sequence on the bicistronic vector pGF121, the vector was constructed. Once completed, pGF120 was electroporated to bacterial strain W3110 and pGF131 was transformed into chemically competent BL21 (DE3) and Lemo21 (De3) Escherichia coli cells. Small-scale expression experiments were carried out as above and compared between bicistronic vectors and dual promoter vectors (pGF119 and pGF120; pGF121 and pGF131).

As shown in Figure 8, the dual T5 promoter produced anti-EGFR1 Fab more efficiently than the bicistronic structure. When using the T7 promoter, the difference was not as obvious, but it was noted that a greater number of unassembled Fab chains appeared when using the dual promoter system than when using the bicistronic structure. The next optimization step planned was to use a chaperone helper plasmid to promote correct folding and assembly in the expression strain. The dual promoter structure with the T7 promoter (vector pGF131) was selected to proceed.

Construction of chaperone helper plasmid

To enhance Fab expression, periplasmic and cytoplasmic chaperones were selected for co-expression with the pGF131 vector. As the backbone vector for the chaperone helper plasmid, pCDF-1b (Novagen) was selected. pCDF-1b has a T7 promoter, a lac operator, a replication origin derived from CloDF13, and streptomycin/spectinomycin antibiotic resistance. It is compatible with co-expression using pET vectors and is suitable for expression with pGF133, which has a pET-15b backbone.

The chaperone protein sequence was amplified from E. coli genomic DNA using PCR and high-fidelity Phusion polymerase (Thermo Scientific). The amplified fragment was cloned into the pCDF-1b backbone using traditional digestion/ligation cloning and seamless Gibson assembly. The structures of the chaperone protein helper plasmids are described in more detail in Tables 18-20.5–7.

Table 18. Cloning strategy for chaperone helper plasmids pGF134, pGF135, pGF137, and pGF138

Table 19. Primer sequences used to construct chaperone helper plasmids.

Table 20. Chaperone proteins used

Anti-EGFR1 Fab co-expression using helper plasmid

According to the manufacturer's instructions, the vector pGF131 was transformed into chemically competent BL21 (DE3) and Lemo21 (DE3) cells. A few clones were selected and the expression of anti-EGFR1 Fab was verified by preliminary expression culture as described above. The best clones were selected as the background for co-expression using the chaperone protein helper plasmid.

Electrocompetent BL21 (De3) pGF131 and Lemo21 (De3) pGF131 cells were constructed as follows. 5 ml pre-culture was cultured overnight in liquid LB supplemented with kanamycin 20 mg/L. On the second day, 1 ml pre-culture was inoculated into 50 ml liquid LB containing kanamycin 20 mg/L. The culture was continued at 220 rpm and 37°C for approximately 3 hours until OD ₆₀₀ reached a level of 0.5. The cells were harvested by centrifugation at 8000 x g for 10 minutes and resuspended in 10 ml 10% ice-cold glycerol. The centrifugation was repeated and the cells were subsequently resuspended in 5 ml 10% ice-cold glycerol. The cells were divided into 10x500 ul aliquots and stored at -80°C.

The chaperone helper plasmids pGF134 and pGF135 were electroporated into BL21(DE3) and Lemo21(DE3) strains using a BioRad Gene Pulser, program Ec2. After a brief pre-culture at +37°C, the mixture was plated onto LB+km+strep and the plates were incubated overnight at +37°C. Primary expression cultures were generated as above.

As exemplified in Figure 9, the SKP chaperone has an obvious beneficial effect on the production process, but is not obvious with the difference of the background strain that only carries expression plasmid pGF131. In any case, clones with the chaperone helper plasmid tend to grow faster and achieve higher cell density. Cultures with the chaperone helper plasmid pGF134 are also more repeatable and more stable. There is no difference between the periplasmic chaperone helper plasmid pGF4134 (SKP chaperone) and pGF135 (SKP and FkpA chaperone). Expression of the cytoplasmic chaperone DnaK/J GrpE from the helper plasmid pGF138 does not further improve expression level. Select its bacterial strains Lemo21 (De3) pGF131 pGF134 and BL21 (De3) pGF131 pGF134 for continuing to develop with fermentation method.

Anti-EGFR single chain

The expression vector pGF155 for the anti-EGFR1 scFv with the signal sequence ompA (SEQ ID NO: 13) was constructed and amplified into the pET-15b backbone using a high-fidelity polymerase and Gibson assembly PCR. In this construct, the polynucleotides encoding the light and heavy chain variable regions are separated by a G4S linker/spacer sequence (SEQ ID NO: 29) encoding the 15-mer linker sequence described in SEQ ID NO: 30.

Vector pGF155 was transformed into the background strain BL21(DE3) alone or in combination with the chaperone helper plasmid, and expression levels were evaluated based on 10 mL preliminary cultures.

Anti-EGFR1 Fab production in fermentation-grown E. coli strains (BL21[DE3]pGF131 pGF134); cultures supplemented with yeast extract.

vaccination

Several (5-8 colonies) of E. coli (BL21[DE3]pGF131 pGF134) from LB agar plates were inoculated into 5 ml of liquid LB medium supplemented with kanamycin (25 mg/L) and streptomycin (30 mg/L). The inoculum (first inoculum) was incubated at +37°C, 220 rpm for 5 hours. 1 ml of the first inoculum was used to inoculate 100 ml of inoculum culture medium (hereinafter) supplemented with kanamycin (25 mg/L) and streptomycin (30 mg/L) in a 500 ml shake flask (second inoculum). The second inoculum was incubated at +37°C, 220 rpm for <16 hours. 10 ml of the second inoculum was transferred into 100 ml of inoculum culture medium (hereinafter) supplemented with kanamycin (25 mg/L) and streptomycin (30 mg/L) in a 500 ml shake flask (third inoculum). The third inoculum was incubated at +37°C, 220 rpm until an _OD600 of approximately 2.0 was reached and this inoculum was used to inoculate 900 ml of fermenter batch culture medium (below) supplemented with kanamycin (25 mg/L) and streptomycin (30 mg/L) in a fermenter broth (2 1) to give a final volume of 1000 ml with an _OD600 value of 0.2.

Table 21. Inoculation medium components (trace metal elements [TME] from FeCl ₃ x6H ₂ O to MgSO ₄ x7H ₂ O).

Reagents Mw (g/mol) mg/l c(mmol/l) 177,99 8600 48,317 174,2 300O 17,222 53,49 1000 18,695 NaCl 58,44 500 8,556 270,33 66 0,245 61,83 3 0,045 161,87 12 0,076 372,24 8,4 0,023 170,48 1,5 0,005 429,89 2,5 0,006 237,93 2,5 0,011 287,54 10 0,036 glucose 180,16 10000 55,506 246,47 600 2,434

Table 22. Fermentation batch culture media (trace metal elements [TME] from FeCl ₃ x 6H ₂ O to MgSO ₄ x 7H ₂ O).

Reagents Mw(g/mol) mg/l c(mmol/l) 174,2 16600 95,293 132,07 4000 30,287 210,14 2297 10,931 270,33 83 0,306 61,83 3,8 0,061 161,87 15 0,095 372,24 10,5 0,028 170,48 1,9 0,011 429,89 3,1 0,007 237,93 3,1 0,013 287,54 13 0,046 glucose 180,16 25000 138,766 246,47 1500 6,086

Fermentation batch stage

After inoculating the fermentation culture tank, set the following parameters using the B Plus digital control unit:

- Temperature +37℃

-pH 6.8 (12.5% NH ₃ , 15% H ₃ PO ₄ )

-pO ₂ (cascade mode)>25%

- Stirring rate 15%-75% (=300 rpm-1500 rpm)

- Air flow (air) 13%-50% (=0.4L-1.5L)

At about 8.5 hours time point in the fermentation batch stage, DOT (dissolved oxygen tension) value violently increases, causes stirring velocity and gas flow to reduce.This shows that the glucose (25g/l) in the batch culture medium exhausts and the fermentation batch stage finishes.During the fermentation batch stage, reach OD ₆₀₀ value 31.

Fed-batch fermentation stage

FS (Feed Solution) 1.1 (67% Glc, 2% _MgSO4 ) was pumped into the fermentation medium at 0.24 mL/min for 6 hours 20 minutes. During this FS1.1 fed-batch phase, an _OD600 value of 70 was reached.

FS 1.2 (50% Glc, 1.5% MgSO ₄ , 7.4 g/100 mL yeast extract, 15-fold TME [trace metal element] concentration compared to the fermentation batch culture medium, 0.32 g/L thiamine) was pumped into the fermentation broth at 0.24 mL/min for 7 hours. An OD ₆₀₀ value of 134 was reached. At this point, the pumping rate was reduced to 0.13 mL/min and continued for 11 hours and 40 minutes. The OD ₆₀₀ value did not increase from 134. Separately, another fermentation was performed without yeast extract supplementation, and this fermentation broth produced approximately 20 mg/L of anti-EGFR1 Fab, estimated by Western blot analysis below.

During the fed-batch phase, the glucose concentration in the culture suspension was followed using Keto-diabur-check 5000 sticks (Roche, cat. no. 10647705187) according to the manufacturer's instructions.

Induction of protein synthesis

Before induction of protein synthesis by IPTG, the culture temperature was lowered from +37°C to +20°C. Protein synthesis was induced by IPTG at an _OD600 value of 86 (final IPTG concentration 1 mM). Induction of protein synthesis was carried out for 16 hours.

Collect samples during fermentation runs

Samples for Western blot analysis were collected at different time points (2 x 1 mL sediment samples and 2 x 1 mL supernatant samples). A pre-induction sample was collected just before IPTG induced protein synthesis. Another set of samples was collected at a 4-hour induction time point. The last set of samples was collected at a 16-hour induction time point before harvesting the culture. The cells in the sample were precipitated (+4°C, 5000 x g for 15 minutes) and the supernatant was transferred to a new tube. The samples were stored at -20°C until Western blot analysis was performed.

Cell harvesting

The fermentation culture suspension was collected in a SLA3000 centrifuge tube (Sorvall RC6) using a Watson Marlow 504U 056.3762.00 pump and the tubes were balanced. The cells were pelleted (+4°C, 5000X g, 60 minutes) and the supernatant discarded. The cell pellet was stored at -20°C.

Western blot analysis of periplasmically expressed anti-EGFR1 Fab

A pellet sample representing 1 mL of fermentation culture suspension was resuspended in 1 mL of 10x TE pH 7.5 (100 mM Tris-HCl, 10 mM EDTA). The sample was vigorously vortexed for 2 hours at room temperature, pelleted at 12,000 x g for 10 minutes at +4°C and the supernatant collected as the periplasmic extract.

The periplasmic extract was further analyzed by Western blotting. 100 μL of the extract was mixed with 25 μL of non-reducing loading buffer. 12.5 μL of the mixture was loaded onto a 4-20% Precise Tris-glycine SDS-Page gel (ThermoScientific). The gel was run at 200V for approximately 45 minutes in 1x Laemmli running buffer and blotted onto a nitrocellulose membrane in Tris-glycine blotting buffer, 350 mA for approximately 1.5 hours. The BioRad Mini-protean system was used for SDS-Page and blotting. The blotted membrane was blocked with 1% BSA in PBS. Detection was performed using anti-human IgG (Fab specific) together with a peroxidase conjugate (Sigma Aldrich; catalog number A0293) and Luminata Forte protein blotting HRP substrate (Millipore; catalog number WBLUF0500). Chemiluminescent reactions were detected using a Fujifilm luminescent image analyzer LAS4000.

10 μL of each culture supernatant sample was mixed with 2.5 μL of non-reducing loading buffer and these samples were electrophoresed in SDS-PAGE gels and blotted onto nitrocellulose membranes as described above for the periplasmic extract samples. The results are shown in FIG10 .

Fab purification

Before the first purification step by means of 5ml cation exchange column (HiTrap SP FF, GE Healthcare), using Amicon Ultra 10K centrifugal filter, the buffer exchange of the periplasmic extract filtered is become 50mM MES pH 6.Mobile phase A is 50mM MES pH 6 and mobile phase B is 50mM MES pH 6+500mM NaCl.Before operation, by 1.2 μm membrane filtration sample.First, 10% sample is injected into this post by flow velocity 2.5ml/ minute for 5 minutes, after which flow velocity is changed to 5ml/ minute.This post is operated with 57.5ml phase A, and applies the linear gradient from 0%B to 100%B in the scope of 35ml subsequently.Collect 2.5mL fractions and gather fraction A5-A9.All the other samples are operated as described above and gather fraction A5-A10 (Figure 11) in two independent tests.The anti-EGFR1 Fab using papain digestion is as control.

Without changing the buffer, the fractions (A5-A10) collected were injected on a protein L column (1ml). Protein L was moved at a flow rate of 0.2ml/min during sample injection and at a flow rate of 1mL/min during washing and elution. Mobile phase A was PBS and mobile phase B was 0.1M sodium citrate at pH 3. The sample was eluted with 100%B. Protein elution had a sharp peak (Figure 12) and fractions A5-A7 were collected and neutralized with 2M Tris-HCl pH 9. After two purification steps, it was estimated that the productive rate of Fab was about 44mg/L. Another batch was only subjected to protein L purification and this produced an approximately 72mg/L Fab fraction. The anti-EGFR1 Fab using papain digestion was used as a control.

The pooled fractions were analyzed in SDS-PAGE. 24 μL of each of the three pooled samples from the chromatography run using a protein L column was mixed with 6 μL of reducing loading buffer and run in an SDS-PAGE gel. The gel was stained with a Coomassie-based stain ( FIG13 ).

Example 14. Binding of anti-EGFR1 Fab and anti-EGFR1 Fab BSH-dextran to EGFR1

Protein A purified anti-EGFR1 produced in CHO cells was digested with papain, purified using a NAb Protein A plus spin column and treated with recombinant Endo F2 (Elizabethkingia meningosepticum) (produced in E. coli, Calbiochem), which cleaves biantennary oligosaccharides and high mannose, leaving a GlcNAc unit on asparagine to obtain a non-glycosylated Fab fragment. 100 mU was added to approximately 1 mg of anti-EGFR1 Fab and incubated overnight at +37°C in 50 mM NaAc pH 4.5.

100 μg anti-EGFR1 Fab and 100 μg anti-EGFR1 Fab BSH-dextran were Cy3-labeled using mono-reactive Amersham Cy3 according to the manufacturer's instructions and 0.5 mg/ml solution was prepared in citrate/phosphate buffer pH 7 to be used for microarray printing.

The array of six different molecules (HER2, human EGFR1, CD64, CD16a, HSA and anti-dextran IgG) was printed on a microarray slide activated by amine-reactive N-hydroxysuccinimide (NHS) (four parallel spots per molecule). Cy3-labeled anti-EGFR1 Fab BSH-dextran conjugates and anti-EGFR1 Fab were incubated at 8 concentrations ranging from 0.4nM to 900nM on the independent wells of the slide. 10x unconjugated BSH dextran was used to remove non-specific binding. After washing the slide, a laser scanner was used to detect the fluorescence signal. The average intensity and standard deviation of each concentration point were calculated from four parallel data points. The following Gmuir isotherm was fitted to the data to calculate K _d value:

F＝(F _max [p])/([p]+K _d )

Where F = fluorescence intensity, _Fmax = maximum intensity at saturation, [p] = concentration of Cy3-labeled molecules and _Kd = dissociation constant.

The anti-EGFR1 Fab BSH-dextran conjugate bound to EGFR1 with a dissociation constant of approximately _Kd = 97 nM. The affinity of the unconjugated Fab for EGFR1 was approximately 2-fold higher than that of the anti-EGFR1 Fab BSH-dextran (Figure 14). Binding of the anti-EGFR1 Fab BSH-dextran or unconjugated Fab to HER2, CD64, CD16a, HSA, or anti-dextran IgG was below the limit of detection.

It is obvious to a person skilled in the art that as technology advances, the basic idea of the invention can be implemented in many ways. The invention and its embodiments are therefore not limited to the examples described above, but they may vary within the scope of the claims.

Sequence Listing

<110> Tengbolong Company

<120> Conjugate

<130> P-WO90874M

<150> FI20155114

<151> 2015-02-20

<150> FI20145552

<151> 2014-06-13

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<170> BiSSAP 1.3

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<211> 1210

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<223> EGF receptor, human NP_005219.2

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Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe

1010 1015 1020

Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala

1025 1030 1035 1040

Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln

1045 1050 1055

Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp

1060 1065 1070

Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro

1075 1080 1085

Val Pro Glu Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser

1090 1095 1100

Val Gln Asn Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser

1105 1110 1115 1120

Arg Asp Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro

1125 1130 1135

Glu Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp

1140 1145 1150

Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp

1155 1160 1165

Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn

1170 1175 1180

Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val

1185 1190 1195 1200

Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210> 2

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain, Cetuximab, INN7906H, from IMGT

<400> 2

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 3

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain, Cetuximab, INN7906L, from IMGT

<400> 3

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 453

<212> PRT

<213> Artificial sequence

<220>

<223> Nimotuzumab_HC

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Asn Pro Thr Ser Gly Gly Ser Asn Phe Asn Glu Lys Phe

50 55 60

Lys Thr Arg Val Thr Ile Thr Val Asp Glu Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys

85 90 95

Ala Arg Gln Gly Leu Trp Phe Asp Ser Asp Gly Arg Gly Phe Asp Phe

100 105 110

Trp Gly Gln Gly Ser Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 5

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> Nimotuzumab_LC

<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asn Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Gln Gln Thr Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Phe Gln Tyr

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr

100 105 110

Arg Glu Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 6

<211> 226

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain Fab

<400> 6

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His

225

<210> 7

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain F(ab')2

<400> 7

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235

<210> 8

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-EGFR1_LC_Variable_DNA

<400> 8

gatattctgc tgacccagtc accggttat ctgagcgtta gtccgggtga acgtgttagc 60

tttagctgtc gtgcaagcca gagcattggc accaatattc attggtatca gcagcgtacc 120

aatggtagtc cgcgtctgct gatcaaatat gcaagcgaaa gcattagcgg tattccgagc 180

cgttttagcg gttctggtag cggcaccgat tttaccctga gtattaatag cgttgaaagc 240

gaagatatcg ccgattatta ctgccagcaa aataacaatt ggccgaccac ctttggtgca 300

ggtacaaaac tggaactgaa ataa 324

<210> 9

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-EGFR1_HC_Variable_DNA

<400> 9

caggtgcagc tgaaacagag cggtccgggt ctggttcagc cgagccagag cctgagcatt 60

acctgtaccg ttagcggttt tagcctgacc aattatggtg ttcattgggt tcgtcagagt 120

ccgggtaaag gtctggaatg gctgggtgtt atttggagcg gtggtaatac cgattataac 180

accccgttta ccagccgtct gagcatcaat aaagataata gcaaaagcca ggtgttcttt 240

aaaatgaata gcctgcagag caatgatacc gccatctatt attgtgcacg tgccctgaca 300

tattatgatt atgaatttgc atattgggga cagggcaccc tggttaccgt tagtgcc 357

<210> 10

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<223> anti-EGFR1 Fab light chain DNA, codon optimized for E. coli

<400> 10

gatattctgc tgacccagag tccggttatt ctgagcgtta gtccgggtga acgtgttagc 60

tttagctgtc gtgcaagcca gagcattggc accaatattc attggtatca gcagcgtacc 120

aatggtagtc cgcgtctgct gatcaaatat gcaagcgaaa gcattagcgg tattccgagc 180

cgttttagcg gtagcggtag tggcaccgat tttaccctga gcattaatag cgttgaaagc 240

gaagatatcg ccgattatta ctgccagcag aacaataatt ggccgaccac ctttggtgca 300

ggtacaaaac tggaactgaa acgtaccgtt gcagcaccga gcgtttttat ctttccgcct 360

agtgatgaac agctgaaaag cggcaccgca agcgttgttt gtctgctgaa taacttttat 420

ccgcgtgaag caaaagttca gtggaaagtt gataatgcac tgcagagcgg taatagccaa 480

gaaagcgtta ccgaacagga tagcaaagat agcacctata gcctgagcag caccctgacc 540

ctgagtaaag cagattatga aaaacacaaa gtgtatgcct gcgaagttac ccatcagggt 600

ctgagcagtc cggtgaccaa aagctttaat cgtggtgaat gttaa 645

<210> 11

<211> 678

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-EGFR1 Fab heavy chain DNA, codon-optimized for E. coli

<400> 11

caggtgcagc tgaagcagtc cggccctggc ctggtgcagc cttcccagtc cctgtccatc 60

acctgtaccg tgtccggctt ctccctgacc aactacggcg tgcactgggt gcgacagtcc 120

cccggcaagg gcctggaatg gctgggagtg atttggagcg gcggcaacac cgactacaac 180

acccccttca cctcccggct gtccatcaac aaggacaact ccaagtccca ggtgttcttc 240

aagatgaact ccctgcagtc caacgacacc gccatctact actgcgccag agccctgacc 300

tactatgact acgagttcgc ctactggggc cagggcaccc tggtgacagt gtccgccgct 360

tccaccaagg gcccctccgt gttccctctg gccccctcca gcaagtccac ctctggcggc 420

accgctgccc tgggctgtct ggtgaaagac tacttccccg agcccgtgac cgtgtcctgg 480

aactctggcg ccctgacctc cggcgtgcac accttccctg ccgtgctgca gtcctccggc 540

ctgtactccc tgtcctccgt ggtgaccgtg ccctccagct ctctgggcac ccagacctac 600

atctgcaacg tgaaccacaa gccctccaac accaaggtgg acaagcgggt ggaacccaag 660

tcctgcgaca agacccac 678

<210> 12

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-EGFR1_scFV_DNA

<400> 12

caggtgcagc tgaaacagag cggtccgggt ctggttcagc cgagccagag cctgagcatt 60

acctgtaccg ttagcggttt tagcctgacc aattatggtg ttcattgggt tcgtcagagt 120

ccgggtaaag gtctggaatg gctgggtgtt atttggagcg gtggtaatac cgattataac 180

accccgttta ccagccgtct gagcatcaat aaagataata gcaaaagcca ggtgttcttt 240

aaaatgaata gcctgcagag caatgatacc gccatctatt attgtgcacg tgccctgaca 300

tattatgatt atgaatttgc atattgggga cagggcaccc tggttaccgt tagtgccggt 360

ggtggtggta gcggtggtgg cggttcaggt ggcggtggtt cagatattct gctgacccag 420

tcaccggtta ttctgagcgt tagtccgggt gaacgtgtta gctttagctg tcgtgcaagc 480

cagagcattg gcaccaatat tcattggtat cagcagcgta ccaatggtag tccgcgtctg 540

ctgatcaaat atgcaagcga aagcattagc ggtattccga gccgttttag cggttctggt 600

agcggcaccg attttaccct gagtattaat agcgttgaaa gcgaagatat cgccgattat 660

tactgccagc aaaataacaa ttggccgacc acctttggtg caggtacaaa actggaactg 720

aaataa 726

<210> 13

<211> 792

<212> DNA

<213> Artificial sequence

<220>

<223> Anti-EGFR1_scFV_DNA_with_ompA

<400> 13

atgaaatacc tgctgccgac cgcagcagcg ggtctgctgc tgctggcagc acagcctgca 60

atggcacagg tgcagctgaa acagagcggt ccgggtctgg ttcagccgag ccagagcctg 120

agcattacct gtaccgttag cggttttagc ctgaccaatt atggtgttca ttgggttcgt 180

cagagtccgg gtaaaggtct ggaatggctg ggtgttattt ggagcggtgg taataccgat 240

tataacaccc cgtttaccag ccgtctgagc atcaataaag ataatagcaa aagccaggtg 300

ttctttaaaa tgaatagcct gcagagcaat gataccgcca tctattattg tgcacgtgcc 360

ctgacatatt atgattatga atttgcatat tggggacagg gcaccctggt taccgttagt 420

gccggtggtg gtggtagcgg tggtggcggt tcaggtggcg gtggttcaga tattctgctg 480

acccagtcac cggttattct gagcgttagt ccgggtgaac gtgttagctt tagctgtcgt 540

gcaagccaga gcattggcac caatattcat tggtatcagc agcgtaccaa tggtagtccg 600

cgtctgctga tcaaatatgc aagcgaaagc attagcggta ttccgagccg ttttagcggt 660

tctggtagcg gcaccgattt taccctgagt attaatagcg ttgaaagcga agatatcgcc 720

gattattact gccagcaaaa taacaattgg ccgaccacct ttggtgcagg tacaaaactg 780

gaactgaaat aa 792

<210> 14

<211> 241

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-EGFR1_scFV

<400> 14

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Leu Leu Thr Gln Ser Pro Val Ile

130 135 140

Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser

145 150 155 160

Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asn Gly

165 170 175

Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser

195 200 205

Ile Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln

210 215 220

Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

225 230 235 240

Lys

<210> 15

<211> 263

<212> PRT

<213> Artificial sequence

<220>

<223> Anti-EGFR1_scFV_with_ompA

<400> 15

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Gln Val Gln Leu Lys Gln Ser Gly Pro Gly

20 25 30

Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly

35 40 45

Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly

50 55 60

Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp

65 70 75 80

Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser

85 90 95

Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr

100 105 110

Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Tyr Asp Tyr Glu Phe

115 120 125

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Leu Leu

145 150 155 160

Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser

165 170 175

Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr

180 185 190

Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser

195 200 205

Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser Glu Asp Ile Ala

225 230 235 240

Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala

245 250 255

Gly Thr Lys Leu Glu Leu Lys

260

<210> 16

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> gIII

<400> 16

Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser

1 5 10 15

His Ser

<210> 17

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> ompA

<400> 17

Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala

1 5 10 15

Thr Val Ala Gln Ala

20

<210> 18

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> malE

<400> 18

Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr

1 5 10 15

Thr Met Met Phe Ser Ala Ser Ala Leu Ala

20 25

<210> 19

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> phoA

<400> 19

Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr

1 5 10 15

Pro Val Thr Lys Ala

20

<210> 20

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> pelB

<400> 20

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala

20

<210> 21

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> stII

<400> 21

Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe Ser

1 5 10 15

Ile Ala Thr Asn Ala Tyr Ala

20

<210> 22

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Internal ribosome binding site

<400> 22

taaggatccg aattcaagga gataaaaaat g 31

<210> 23

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Primer sequences used to construct GP1113

<400> 23

cgggatccaa gaaggagata taccatggca aaaaagtggt tattagctgc 50

<210> 24

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Primer sequences used to construct GP1114

<400> 24

ataatgcggc cgcattattt aacctgtttc agtac 35

<210> 25

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> Primer sequences used to construct GP1115

<400> 25

ataatgcggc cgcaagaagg agatatacca tggcaaaatc actgtttaaa gtaacg 56

<210> 26

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Primer sequences used to construct GP1116

<400> 26

ataatctcga gattattttt tagcagaatc tgc 33

<210> 27

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Primer sequences used to construct GP1147

<400> 27

tgacccgcta atgcggccgc actgagtgct tcccttgaaa ccctgaaact gatc 54

<210> 28

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Primer sequences used to construct GP1148

<400> 28

ggtttcttta ccagactcaa acggcccggc attcgcatgc agggccgtga attattacg 59

<210> 29

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> G4S_Linker/Spacer_DNA

<400> 29

ggtggtggtg gtagcggtgg tggcggttca ggtggcggtg gttca 45

<210> 30

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> G4S_Linker/Spacer

<400> 30

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Claims (73)

1. A conjugate comprising an anti-EGFR1 antibody or an EGFR1-binding fragment thereof and at least one dextran derivative, wherein The dextran derivative comprises at least one D-glucopyranosyl unit, wherein at least one carbon selected from carbon 2, 3, or 4 of the at least one D-glucopyranosyl unit is substituted by a substituent of the following formula. ^-O- _{( CH₂} ) _{nSB₁₂H₁₁₂-} Where n is in the range of 3 to 10; and The dextran derivative binds to the anti-EGFR antibody or its EGFR1 binding fragment via a bond formed by a reaction between at least one aldehyde group and an amino group of the anti-EGFR1 antibody or its EGFR1 binding fragment, wherein the at least one aldehyde group is formed by oxidative cleavage of the D-glucopyranosyl unit of the dextran derivative. 2. The conjugate according to claim 1, wherein the molecular weight of the dextran derivative is in the range of 3 to 2000 kDa. 3. The conjugate according to claim 1, wherein the molecular weight of the dextran derivative is in the range of 30 to 300 kDa. 4. The conjugate according to claim 1, wherein the conjugate comprises 10 to 300 substituents ^of the formula -O-( _CH2 ) _{n- SB12H112- .} 5. The conjugate according to claim 1, wherein the conjugate comprises 20 to 150 substituents ^of the formula -O-( _CH2 ) _{n -SB12H112- .} 6. The conjugate according to any one of claims 1–5, wherein the amino group of the anti-EGFR1 antibody or its EGFR1 binding fragment is an amino group of a lysine residue of the anti-EGFR1 antibody or its EGFR1 binding fragment. 7. The conjugate according to any one of claims 1–5, wherein the conjugate further comprises at least one tracer molecule that binds to the dextran derivative or to the anti-EGFR1 antibody or its EGFR1 binding fragment. 8. The conjugate according to any one of claims 1–5, wherein the dextran derivative comprises at least one aldehyde group capped by oxidative cleavage of the D-glucopyranosyl unit of the dextran derivative. 9. The conjugate of claim 8, wherein the dextran derivative comprises a plurality of aldehyde groups formed by the oxidative cleavage of the D-glucopyranosyl unit of the dextran derivative, and the aldehyde groups formed by the oxidative cleavage of one or more D-glucopyranosyl units of the dextran derivative are substantially all end-capped. 10. The conjugate according to any one of claims 1–5, which can be obtained by a method comprising the following steps: a) Alkenylate at least one hydroxyl group of dextran to obtain alkenylated dextran; b) Reaction of sodium borate with alkenylated dextran obtained from step a) to obtain BSH-dextran; c) Oxidatively cleaving at least one D-glucopyranosyl residue of BSH-glucan to form an aldehyde group; d) The oxidatively cleaved BSH-glucan obtained from step c) is reacted with an anti-EGFR1 antibody or its EGFR1-binding fragment to obtain a conjugate; In BSH-glucan, BSH stands for sodium mercaptododecanoate or thioborane. 11. The conjugate according to claim 10, wherein the dextran is alkenylated in step a) using an alkenylating agent, wherein the alkenylating agent has the structure of the following formula. X-( _CH₂ ) _mCH ＝ _CH₂ Where m is in the range of 1 to 8, and X is Br, Cl or I. 12. The conjugate according to claim 10, wherein at least one carbon selected from carbon 2, 3, or 4 of the alkenylated dextran obtainable from step a) is substituted by a substituent of the following formula. -O-( _CH₂ ) _mCH ＝ _CH₂ , Where m is in the range of 1 to 8. 13. The conjugate according to claim 10, wherein sodium borate and the alkenylated dextran obtainable from step a) are reacted in step b) in the presence of a free radical initiator selected from ammonium persulfate, potassium persulfate and ultraviolet light. 14. The conjugate according to claim 10, wherein the at least one D-glucanyl residue of the BSH-glucan is oxidatively cleaved in step c) using an oxidant selected from sodium periodate, periodic acid and lead acetate (IV). 15. The conjugate according to claim 10, wherein the method further comprises the step of reacting the oxidically cleaved BSH-glucan available from step c) or the conjugate available from step d) with a tracer molecule. 16. The conjugate according to claim 10, wherein the method further comprises step e): capping an unreacted aldehyde group of the oxidatively cleaved BSH-glucan available in step c) or the conjugate available in step d). 17. The conjugate according to claim 16, wherein the unreacted aldehyde group is capped with a hydrophilic capping agent such as ethanolamine, lysine, glycine, or Tris. 18. The conjugate according to claim 10, wherein the molecular weight of the dextran is in the range of 3 to 2000 kDa. 19. The conjugate according to claim 10, wherein the molecular weight of the dextran is in the range of 10 to 100 kDa. 20. The conjugate according to claim 10, wherein the molecular weight of the dextran is in the range of 5 to 200 kDa. 21. The conjugate according to claim 10, wherein the molecular weight of the dextran is in the range of 10 to 250 kDa. 22. The conjugate according to claim 10, wherein the oxidically cleaved BSH-glucan is reacted with the anti-EGFR1 antibody or its EGFR1 binding fragment by incubating the oxidically cleaved BSH-glucan and the anti-EGFR1 antibody or its EGFR1 binding fragment at room temperature in step d) in an aqueous phosphate buffer solution having a pH of 6 to 8. 23. The conjugate according to any one of claims 11-21, wherein the oxidically cleaved BSH-glucan is reacted with the anti-EGFR1 antibody or its EGFR1 binding fragment by incubating the oxidically cleaved BSH-glucan and the anti-EGFR1 antibody or its EGFR1 binding fragment in step d) at room temperature in an aqueous phosphate buffer solution having a pH of 6 to 8. 24. A pharmaceutical composition comprising the conjugate according to any one of claims 1-23. 25. The pharmaceutical composition according to claim 24, for treating cancer. 26. The pharmaceutical composition of claim 25, wherein the cancer is head and neck cancer. 27. The conjugate according to any one of claims 1-5, 9, 11-22, for the treatment of cancer. 28. The conjugate according to claim 27, wherein the cancer is head and neck cancer. 29. Use of the conjugate of any one of claims 1-23 or the pharmaceutical composition of claim 24 in the preparation of a medicament for treating or regulating the growth of tumor cells expressing EGFR1 in humans. 30. The use according to claim 29, wherein the conjugate or the pharmaceutical composition is administered intratumorally and/or intravenously. 31. The use according to claim 29, wherein the concentration of boron in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 1:1. 32. The use according to claim 29, wherein the boron concentration in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 2:1. 33. The use according to claim 29, wherein the concentration of boron in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 3:1. 34. The use according to claim 29, wherein the concentration of boron in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 4:1. 35. The use according to claim 29, wherein the concentration of boron in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 5:1. 36. The use according to claim 29, wherein the boron concentration in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 6:1. 37. The use according to claim 29, wherein the concentration of boron in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 7:1. 38. The use according to claim 29, wherein the boron concentration in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 8:1. 39. The use according to claim 29, wherein the concentration of boron in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 10:1. 40. The use according to claim 29, wherein the concentration of boron in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 13:1. 41. The use according to claim 29, wherein the boron concentration in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 130:1. 42. The use according to claim 29, wherein the boron concentration in tumor cells and blood is analyzed after administration of the conjugate or the pharmaceutical composition, and the ratio of boron concentration in tumor cells to boron concentration in blood is greater than 240:1. 43. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition is used for intratumoral and/or intravenous treatment of head and neck cancer via boron neutron capture therapy. 44. The conjugate according to any one of claims 1-5, 9, 11-22, wherein the anti-EGFR1 antibody or its EGFR1-binding fragment is obtained by a method comprising: Culture prokaryotic host cells containing one or more polynucleotides encoding i) a light chain variable region and ii) a heavy chain variable region encoding an anti-EGFR1 antibody or its EGFR1-binding fragment; and Isolate and/or purify the anti-EGFR1 antibody or its EGFR1-binding fragment. 45. The conjugate according to claim 44, wherein the host cell is an Escherichia coli cell. 46. The conjugate of claim 44, wherein the one or more polynucleotides encoding the light chain variable region and the heavy chain variable region are codon-optimized for the host cell. 47. The conjugate of claim 44, wherein the host cell comprises a single continuous polynucleotide encoding a light chain variable region and a heavy chain variable region of the anti-EGFR1 antibody or an EGFR1 binding fragment thereof. 48. The conjugate of claim 44, wherein the host cell comprises a polynucleotide encoding a light chain variable region encoding an anti-EGFR1 antibody or an EGFR1-binding fragment thereof and another polynucleotide encoding a heavy chain variable region encoding an anti-EGFR1 antibody or an EGFR1-binding fragment thereof. 49. The conjugate according to claim 44, wherein a signal peptide precedes the light chain variable region and the heavy chain variable region. 50. The conjugate according to claim 49, wherein the signal peptide preceding the variable region of the light chain is different from the signal peptide preceding the variable region of the heavy chain. 51. The conjugate according to claim 49 or 50, wherein the light chain variable region and the signal peptide preceding the heavy chain variable region are independently selected from gIII, malE, phoA, ompA, pelB, and stII. 52. The conjugate according to claim 49 or 50, wherein the light chain variable region and the signal peptide preceding the heavy chain variable region are independently selected from ompA, pelB, and stII. 53. The conjugate according to claim 49, wherein the signal peptide preceding the variable region of the light chain is the same as the signal peptide preceding the variable region of the heavy chain, and wherein the signal peptide is selected from gIII, malE, phoA, ompA, pelB, and stII. 54. The conjugate according to claim 49, wherein the signal peptide preceding the variable region of the light chain is the same as the signal peptide preceding the variable region of the heavy chain, and wherein the signal peptide is selected from ompA, pelB, and stII. 55. The conjugate according to claim 49 or 50, wherein the light chain variable region is preceded by a pelB signal peptide and the heavy chain variable region is preceded by an ompA signal peptide. 56. The conjugate according to claim 49, wherein both the light chain variable region and the heavy chain variable region are preceded by an stII signal peptide. 57. The conjugate according to claim 44, wherein the polynucleotide encoding the light chain variable region comprises or is composed of the sequence described in or at least 99% identical to the sequence in SEQ ID NO:8, and the polynucleotide encoding the heavy chain variable region comprises or is composed of the sequence described in or at least 99% identical to the sequence in SEQ ID NO:9. 58. The conjugate of claim 44, wherein the host cell comprises one or more polynucleotides encoding an anti-EGFR1 binding fragment of the antibody. i) Light chains and ii) Heavy chains. 59. The conjugate of claim 44, wherein the one or more polynucleotides encode an anti-EGFR1 binding fragment as Fab or scFv. 60. The conjugate of claim 44, wherein the one or more polynucleotides comprise, or consist of, the light chain sequence described in SEQ ID NO:10 or at least 99% identical to the sequence described in SEQ ID NO:10, and the heavy chain sequence described in SEQ ID NO:11 or at least 99% identical to the sequence described in SEQ ID NO:11. 61. The conjugate of claim 44, wherein the host cell comprises a polynucleotide, the polynucleotide comprising or consisting of the sequence described in SEQ ID NO:12 or a sequence that is at least 99% identical to or constitutes the sequence described in SEQ ID NO:12. 62. The conjugate of claim 44, wherein the host cell comprises a polynucleotide, the polynucleotide comprising or consisting of the sequence described in SEQ ID NO:13 or a sequence that is at least 99% identical to or constitutes the sequence described in SEQ ID NO:13. 63. The conjugate of claim 44, wherein the host cell comprises a chaperone protein and/or one or more polynucleotides encoding a chaperone protein. 64. The conjugate according to claim 63, wherein the chaperone protein is selected from DnaK, DnaJ, GrpE, Skp, FkpA, GroEL, and GroES. 65. The conjugate according to claim 63 or 64, wherein the chaperone protein is Skp. 66. The conjugate according to claim 44, wherein the host cell lacks one or more proteolytic enzymes. 67. The conjugate according to claim 66, wherein the proteolytic enzyme is selected from protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and Lon. 68. The conjugate of claim 44, wherein the one or more polynucleotides are driven by promoters independently selected from T7, T5 and Rham. 69. The conjugate of claim 44, wherein the one or more polynucleotides are driven by promoter T7. 70. The conjugate of claim 44, wherein the light chain variable region is preceded by a pelB signal peptide and the heavy chain variable region is preceded by an ompA signal peptide, the host cell contains the chaperone protein Skp and/or a polynucleotide encoding the chaperone protein Skp; and the host cell lacks the proteolytic enzymes Lon and OmpT. 71. The conjugate of claim 44, wherein the light chain variable region and the heavy chain variable region are preceded by an stII signal peptide, the host cell contains the chaperone protein Skp and/or a polynucleotide encoding the chaperone protein Skp; and the host cell lacks the proteolytic enzymes Lon and OmpT. 72. The conjugate according to claim 44, wherein the anti-EGFR1 antibody or its EGFR1 binding fragment comprises or is composed of the amino acid sequence described in SEQ ID NO:14 or SEQ ID NO:15. 73. Use of the conjugate according to claim 44 in the preparation of a medicament for treating or regulating the growth of tumor cells expressing EGFR1 in humans.