CN112375140A - Rapid and large-flux rabbit polyclonal antibody in-vitro production method - Google Patents
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Abstract
The invention provides a rapid and large-flux rabbit polyclonal antibody in-vitro production method, which mainly utilizes a B lymphocyte separation technology, realizes the rapid, large-scale and stable production of the rabbit polyclonal antibody through in-vitro amplification culture, optimizes a B lymphocyte culture medium, adopts tryptone and matches with blank rabbit serum to prepare the B lymphocyte culture medium, greatly improves the differentiation and proliferation amount of B lymphocytes, and provides a basis of cell amount for the in-vitro production of the rabbit polyclonal antibody. By the method provided by the invention, the rabbit can be produced by blood collection immediately after the third immunization, the production period of the product is greatly shortened, the yield of the obtained rabbit polyclonal antibody is more than 30 times of the yield of the same volume of serum in the traditional method, and the method has the advantages of short period, high yield, low unit cost, stable quality and the like and is suitable for large-scale production and application.
Description
Technical Field
The invention relates to the field of antibody preparation, in particular to a rapid and high-flux rabbit polyclonal antibody in-vitro production method.
Background
The rabbit polyclonal antibody refers to an antibody generated by the immune system of rabbits due to the stimulation of an antigen. Often, the antigen is composed of multiple epitopes, and a rabbit B lymphocyte can only be stimulated by one epitope of the antigen to produce a corresponding specific antibody, which is called a monoclonal antibody. Therefore, the various antigenic determinants stimulate the immune system of rabbits, and accordingly, various specific monoclonal antibodies are produced, and the monoclonal antibodies are mixed together to form polyclonal antibodies. The antibody generated by the rabbit immune system has the advantages of strong affinity, good specificity and high titer for other species such as mice, rats and the like, so that the rabbit polyclonal antibody provides an optimal antibody reagent source for scientific research and development, drug screening and clinical diagnosis.
The in vitro production of the antibody refers to the in vitro culture of cells or tissues secreting the antibody by using a specific artificial environment and culture reagents to obtain the specific antibody. The common antibody in vitro culture techniques include hybridoma cell culture technique, recombinant antibody culture technique, etc., but such techniques are often used for monoclonal antibody production.
The traditional rabbit polyclonal antibody production method is mainly characterized in that the rabbit is immunized in a large scale by using antigen, then 20-30ml of rabbit serum is collected after the fourth immunization, and then the blood is taken, wherein the circulation cycle of immunization, rest, blood collection and rest is required to be repeated. Thus it takes 105 days if 100ml of rabbit serum is collected. And because the health, the life-span of the rabbit are restricted, the polyclonal antibody product often needs to immunize a new rabbit again, which causes the problems of large batch difference and unstable quality of the product.
Disclosure of Invention
In order to solve the problems, the invention provides a method for realizing the rapid, large-scale and stable production of rabbit polyclonal antibodies by using a B lymphocyte separation technology through in-vitro amplification culture, optimizes a B lymphocyte culture medium, adopts tryptone and matches blank rabbit serum as a special additive, greatly improves the differentiation and proliferation amount of the B lymphocytes, and provides a basis of cell amount for the in-vitro production of the rabbit polyclonal antibodies. By the method provided by the invention, the rabbit can be produced by blood collection immediately after the third immunization, the production period of the product is greatly shortened, the yield of the obtained rabbit polyclonal antibody is more than 30 times of the yield of the same volume of serum in the traditional method, and the method has the advantages of short period, high yield, low unit cost, stable quality and the like and is suitable for large-scale production and application.
In one aspect, the invention provides a rapid, large-flux rabbit polyclonal antibody in vitro production method, which is characterized by comprising the following steps:
1) obtaining peripheral blood mononuclear cell suspension on the immunized rabbit;
2) obtaining antigen specific B lymphocytes by using a high affinity plate coated with antigen;
3) obtaining an optimized B lymphocyte culture medium;
4) adopting the B lymphocyte culture medium optimized in the step 3) to culture specific B lymphocytes in an expanding way;
5) and (5) affinity purifying the supernatant to quickly obtain a large amount of rabbit polyclonal antibody products.
Further, the optimized B lymphocyte culture medium in step 3) is: RPMI1640+ 10% blank rabbit serum + 1% tryptone + additive A, said additive A is an important cytokine for substituting Th cell to activate B lymphocyte.
The existing B cell culture medium is RPMI1640 containing 10% FBS, and additive A (additive A is an important cytokine for substituting Th cell to activate B cell, such as CD40L and IL6, etc.) is added, and the additive A preferably used in the invention is CD40L and IL 6.
The optimized B cell culture medium comprises the following components: RPMI1640+ 10% blank rabbit serum + 1% tryptone + additive a. The tryptone can better provide nutrition for synthesizing the antibody as a nitrogen source, and the blank rabbit serum can better stimulate the division and proliferation of the rabbit B cells as an additive rich in rabbit-derived cytokines.
B cells need to consume a large amount of amino acids to synthesize proteins when they secrete antibodies, and therefore a nitrogen source is additionally added as a supplement. The most commonly used method in cell culture is MEM Amino Acids and MEM NEAA, which are directly supplemented with Amino Acids, and the method has high cost, and the rabbit B cell consumes undefined proportion of Amino Acids, which may not achieve the optimal effect. Tryptone is frequently used as a nitrogen source in bacterial culture, and is rarely used in cell culture. We have found that tryptone is a particularly effective and inexpensive nitrogen source supplement for B cell culture.
In addition, in the process of optimizing the culture medium, considering that the rabbit B lymphocyte is required to secrete a large amount of antibody for producing rabbit polyclonal antibody, the species difference of the cytokine in serum is reduced by replacing FBS (fetal bovine serum) with rabbit serum in terms of the cytokine, and the B cell is stimulated and activated better.
The invention optimizes the culture condition of the B lymphocyte, takes tryptone and blank rabbit serum as special additives, optimizes and improves the conventional culture medium, greatly improves the differentiation and proliferation amount of the B lymphocyte, and provides a basis of cell amount for the in vitro production of rabbit polyclonal antibodies.
Further, the peripheral blood of the rabbit obtained after the third immunization in the step 1) is used for preparing the peripheral blood mononuclear cell suspension.
Further, the preparation method of the peripheral blood mononuclear cell suspension in the step 1) comprises the following steps:
a) adding 20mL of lymphocyte separation solution Ficoll into a 50mL centrifuge tube;
b) taking 10mL of third-immunized anticoagulated rabbit venous blood and 10mL of sterile DPBS according to the ratio of 1:1, fully and uniformly mixing, and slowly overlapping on the layered liquid level along the tube wall by using a pipette;
c) horizontal centrifugation is carried out for 400g multiplied by 30 minutes;
d) peripheral Blood Mononuclear Cells (PBMCs) were pipetted into a 50ml centrifuge tube;
e) adding DPBS to a constant volume of 45ml, centrifuging for 300g multiplied by 10 minutes, and washing cells twice by using the DPBS under the same condition;
f) after the last centrifugation, the supernatant was discarded, RPMI1640 containing 10% FBS was added, the cells were resuspended, and counted.
Further, the coated antigen in the step 2) is immunogen protein, polypeptide of cross-linked KLH or BSA.
Further, the method for obtaining antigen-specific B lymphocytes by using the high affinity plate coated with antigen in step 2) comprises:
i) filtering the antigen by using a 0.22 mu m filter element, coating a high affinity plate with the filtered antigen, and coating overnight at 4 ℃;
II) the next day, the supernatant was blotted and washed twice with 10ml PBS, blocked with 5% FBS/PBS, and blocked at room temperature for 2 hours or at 37 ℃ for 30 min;
III) adding the peripheral blood mononuclear cells separated in the step f) in the step 1) into a high affinity plate according to the amount of 5M/plate, and incubating for one and half hours at 37 ℃;
IV) washing the incubated plate with RPMI1640 for 3 times;
v) collecting the separated cells.
Further, the conditions of the scale-up culture in the step 4) are as follows: antigen-specific B lymphocytes were cultured in a dish at 37 ℃ in a 5% CO2 incubator for 5 days, then transferred to a conical flask, further cultured in a 37 ℃ 5% CO2 shaking incubator for 15 days, and centrifuged to collect the supernatant.
Further, the affinity purification method in step 5) comprises the following steps: affinity chromatography was performed using an antigen affinity column.
In another aspect, the present invention provides a B lymphocyte culture medium having tryptone as a nitrogen source.
Further, the B lymphocyte culture medium is: RPMI1640+ 10% blank rabbit serum + 1% tryptone + additive A, said additive A is important cytokine for substituting Th cell to activate B cell, such as cytokine of CD40L, IL6, etc., additive A preferably used in the invention is CD40L and IL 6.
Further, the B lymphocyte is an antigen-specific B lymphocyte derived from a rabbit peripheral blood mononuclear cell suspension.
In a further aspect, the invention provides the use of tryptone for the preparation of a culture medium for B lymphocytes.
Further, the B lymphocyte culture medium is: RPMI1640+ 10% blank rabbit serum + 1% tryptone + additive A, said additive A is important cytokine for substituting Th cell to activate B cell, such as cytokine of CD40L, IL6, etc., additive A preferably used in the invention is CD40L and IL 6.
Further, the B lymphocyte is an antigen-specific B lymphocyte derived from a rabbit peripheral blood mononuclear cell suspension.
The rapid and large-flux rabbit polyclonal antibody in-vitro production method provided by the invention has the following beneficial effects:
1. when the method provided by the invention is adopted to produce rabbit polyclonal antibodies, rabbits can take blood for production immediately after the third immunization, the production cycle is greatly shortened, and the production cycle of 100mg per product can be shortened by half;
2. by optimizing a B lymphocyte culture medium and adopting tryptone and blank rabbit serum as special additives, the differentiation and proliferation amount of the B lymphocyte is greatly improved, and a basis of cell amount is provided for the in vitro production of rabbit polyclonal antibodies.
3. The yield of the rabbit polyclonal antibody obtained by the serum with the same volume is more than 30 times of that obtained by the traditional method;
4. has the advantages of short period, large yield, low unit cost, stable quality and the like, and is suitable for large-scale production and application.
Drawings
FIG. 1 is a flow chart of the method for the in vitro production of rabbit polyclonal antibodies with high speed and high flux as provided in example 1
FIG. 2 is a graph showing the growth of B lymphocytes cultured in different media according to example 2
FIG. 3 is a 10-fold dilution of the cell density observed after culturing B lymphocytes in different media for 9 days in example 2
FIG. 4 is a graph showing the immunofluorescence staining results of anti-rabbit IgG in example 2 in which B lymphocytes were cultured in different media
FIG. 5 shows the product quality obtained by WB assay according to example 3, wherein channel 1 is the product purified according to the method provided in example 1, channel 2 is the product purified according to conventional serum, channel 3 is a negative control, channel 4 is a positive control
Detailed Description
In the following, preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings, it being noted that the following embodiments are intended to facilitate understanding of the present invention without any limitation thereto.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.
Example 1 Rapid, Large-throughput in vitro production of Rabbit polyclonal antibodies
The method for producing rabbit polyclonal antibody in vitro with rapid and large flux, which is provided by the embodiment, is shown in the invention flow chart (figure 1), and comprises the following steps:
step 1): obtaining peripheral blood mononuclear cell suspension from immunized rabbit
In this embodiment, it is preferable that the peripheral blood mononuclear cell suspension is obtained from a rabbit after the third immunization, and the preparation method of the peripheral blood mononuclear cell suspension comprises:
a) 20mL of lymphocyte separation solution Ficoll (Tianjin plus sunlight, Cat #: LTS 1077);
b) 10mL of anticoagulated rabbit venous blood after the third immunization was mixed with 10mL of sterile DPBS (Gibco, Cat #: d8537-500ML) according to 1:1, fully and uniformly mixing, and slowly overlapping on the layered liquid level along the tube wall by using a pipette;
c) horizontal centrifugation is carried out for 400g multiplied by 30 minutes;
d) peripheral Blood Mononuclear Cells (PBMCs) were pipetted into a 50ml centrifuge tube;
e) adding DPBS to a constant volume of 45ml, centrifuging for 300g multiplied by 10 minutes, and washing cells twice by using the DPBS under the same condition;
f) after the final centrifugation, the supernatant was discarded, and a suspension containing 10% FBS (Hyclone, Cat #: SV30208) RPMI1640(Gibco, Cat #: c11875500BT), resuspend the cells, count.
Step 2) obtaining antigen specific B lymphocytes by using high affinity plate coated with antigen
The method for obtaining antigen-specific B lymphocytes by using the high affinity plate coated with the antigen is as follows:
i) filtering the antigen by using a 0.22 mu m filter element, coating a high affinity plate with the filtered antigen, and coating overnight at 4 ℃;
II) the next day, the supernatant was blotted and washed twice with 10ml PBS, blocked with 5% FBS/PBS, and blocked at room temperature for 2 hours or at 37 ℃ for 30 min;
III) adding the peripheral blood mononuclear cells separated in the step f) in the step 1) into a high affinity plate according to the amount of 5M/plate, and incubating for one and half hours at 37 ℃;
IV) the incubated plate was washed 3 times with RPMI 1640.
Step 3) obtaining optimized B lymphocyte culture medium
The method for optimizing the B lymphocyte culture medium comprises the following steps: and (2) respectively taking 10ml of the traditional B lymphocyte culture medium and a plurality of groups of optimized culture media with different components, inoculating the antigen-specific B lymphocyte obtained in the step (2) into a dish, controlling the cell density to be 0.01M/ml, continuously culturing for 15 days, and counting every 2 days to draw a cell growth curve.
The B lymphocyte culture medium obtained by optimization is as follows: RPMI1640+ 10% blank rabbit serum (from the animal house at university of Hangzhou university) + 1% tryptone + additive A, which is an important cytokine to replace Th cell activated B lymphocytes, the additive A in this example is preferably CD40L (from abcam Cat #: ab179625) and IL6 (from Noveprotein, Cat #: CG 39).
Step 4) adopting the B lymphocyte culture medium optimized in the step 3) to enlarge and culture the antigen specific B lymphocyte
The main method comprises the following steps: antigen-specific B lymphocytes were cultured in the optimized B lymphocyte medium, first cultured in a dish in a 37 ℃ 5% CO2 incubator for 5 days, then transferred to a conical flask, further cultured in a 37 ℃ 5% CO2 shake flask for 15 days, centrifuged at 800 g.times.20 minutes, and the culture supernatant was collected.
Step 5) affinity purifying the supernatant to quickly obtain a large amount of rabbit polyclonal antibody products
The method mainly adopts an antigen affinity column for affinity chromatography, and comprises the following steps:
mixing the collected supernatant with PBS according to the volume (1:1), centrifuging at 8000rpm at room temperature for 15 min; the diluted sample flows through the affinity column at a speed of 10-30ml/h, the flow-through liquid is received by a centrifugal tube, and the operation is repeated for 1-2 times;
eluting the antibody from the affinity column by eluent, and neutralizing the PH value to 7-8 to obtain a final polyclonal antibody product;
thirdly, the nanodrop instrument is utilized to measure the concentration of the antibody and calculate the yield of the antibody.
Example 2 optimization of B lymphocyte culture media
The method for optimizing the B lymphocyte culture medium comprises the following steps: and (2) respectively taking 10ml of the traditional B lymphocyte culture medium and a plurality of groups of optimized culture media with different components, inoculating the antigen-specific B lymphocyte obtained in the step (2) into a dish, controlling the cell density to be 0.01M/ml, continuously culturing for 15 days, and counting every 2 days to draw a cell growth curve.
Wherein, the traditional B lymphocyte culture medium is a control group, and a plurality of groups of culture media with different components for optimization are test groups:
control group: b cell conventional medium formula: RPMI1640(Gibco, Cat #: C11875500BT) + CD40L (abcam, Cat #: ab179625) +20ng/ml IL-6(Noveprotein, Cat #: CG39) + 10% FBS (Hyclone, Cat #: SV 30208);
test group 1: RPMI1640(Gibco, Cat #: C11875500BT) + CD40L (abcam, Cat #: ab179625) +20ng/ml IL-6(Noveprotein, Cat #: CG39) + 10% blank rabbit serum (purchased from Hangzhou Master and university animal house)
Test group 2: RPMI1640(Gibco, Cat #: C11875500BT) + CD40L (abcam, Cat #: ab179625) +20ng/ml IL-6(Noveprotein, Cat #: CG39) + 10% FBS (Hyclone, Cat #: SV30208) + 1% tryptone
Test group 3: RPMI1640(Gibco, Cat #: C11875500BT) + CD40L (abcam, Cat #: ab179625) +20ng/ml IL-6(Noveprotein, Cat #: CG39) + 10% blank rabbit serum (purchased from animal houses of Hangzhou Master and Fan university) +1X MEM Amino Acids (Gibco, Cat #11130051) +1X MEM NEAA (Gibco, Cat #: 11140050)
Test group 4: RPMI1640(Gibco, Cat #: C11875500BT) + CD40L (abcam, Cat #: ab179625) +20ng/ml IL-6(Noveprotein, Cat #: CG39) + 10% white rabbit serum (purchased from the animal house of the university of Hangzhou Master) + 1% tryptone.
The B lymphocyte culture growth curve is shown in figure 2; at day 9 of culture, the cells were diluted 10-fold to observe the cell density, and the results are shown in FIG. 3, in which FIG. 3A is a control group, FIG. 3B is a test group 1, FIG. 3C is a test group 2, FIG. 3D is a test group 3, and FIG. 3E is a test group 4; the optimal test group 4 was taken and stained by anti-rabbit IgG immunofluorescence, and the results are shown in fig. 4.
As can be seen from fig. 2, by comparing the growth curves of the B cells after culture, the optimized culture medium of test group 4 can greatly improve the cell proliferation capacity; as can be seen from fig. 3, the cell density of the test group 4 was 5 times higher than that of the control group at the 9 th day of culture; from the results of immunofluorescence staining with anti-rabbit IgG in fig. 4, all proliferating cells were B lymphocytes secreting rabbit IgG. The optimized culture medium (test group 4) can greatly improve the differentiation and proliferation amount of the rabbit B lymphocyte, and provides a basis for the cell amount for the production of polyclonal antibodies.
The optimized medium of test group 4 was supplemented with tryptone as a nitrogen source in addition to the control group. The research group finds that a large amount of Amino acid is consumed to synthesize protein when B cells secrete antibodies, so a nitrogen source needs to be supplemented, MEM Amino Acids and MEM NEAA are the most commonly used in cell culture, Amino acid is directly supplemented, but the method is high in cost, the proportion of Amino acid consumed by the B cells of rabbits is unclear, the best effect cannot be achieved, tryptone is frequently used as a nitrogen source in bacterial culture and is rarely used in cell culture, and the research group creatively adopts the tryptone as the nitrogen source to achieve a very good culture effect. In addition, the species differences of cytokines in serum were reduced by substituting blank rabbit serum for FBS (fetal bovine serum), better stimulating and activating B cells.
This embodiment is through regard as rabbit serum and tryptone as special additive, optimized the improvement to conventional culture medium, has obtained the B lymphocyte culture condition of optimizing, and the cell density peak value is 5 times before optimizing, and almost all cells all are the B lymphocyte of secretion antibody, promote rabbit B lymphocyte's expansion reproductive capacity by a wide margin, and the optimization condition is very effective, and the effect is showing.
Example 3 comparison with conventional Process
In this example, the in vitro rabbit polyclonal antibody production method provided in example 1 (wherein the B lymphocyte culture medium employs the test group 4 optimized in example 2) and the traditional production method are respectively used to compare the in vitro rabbit polyclonal antibody production effects, wherein the traditional production method is to collect rabbit serum after the fourth immunization, the results are shown in table 1, and the product quality obtained by WB detection is shown in fig. 5, wherein channel 1 is the product purified by the method provided in example 1, channel 2 is the product purified by the traditional serum, channel 3 is a negative control, and channel 4 is a positive control.
TABLE 1 comparison of the Effect of the method provided in example 1 on the in vitro production of rabbit polyclonal antibodies with the conventional method
As can be seen from table 1, the method provided in example 1 produces more than 30 times the yield of the same volume of serum; as can be seen from fig. 5, the quality of the product obtained by the WB assay using the method provided in example 1 was the same as that obtained by the conventional serum production method.
In addition, the technical scheme provided by the embodiment 1 is only limited to the optimization of the in vitro culture conditions of the specific B lymphocytes from the peripheral blood of the rabbits to produce the rabbit polyclonal antibody, and compared with the traditional rabbit blood collection polyclonal antibody production method, the method provided by the embodiment 1 can be used for collecting blood immediately after the third immunization to produce the rabbit polyclonal antibody (the production period for producing 100mg can be shortened by half), and has the advantages of short period, large yield, low unit cost, stable quality and the like.
Although the present invention is disclosed above, the present invention is not limited thereto. For example, the application range of the micro-fluidic field can be expanded. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A rapid and large-flux rabbit polyclonal antibody in vitro production method is characterized by comprising the following steps:
1) obtaining peripheral blood mononuclear cell suspension on the immunized rabbit;
2) obtaining antigen specific B lymphocytes by using a high affinity plate coated with antigen;
3) obtaining an optimized B lymphocyte culture medium;
4) adopting the B lymphocyte culture medium optimized in the step 3) to culture antigen specific B lymphocytes in an enlarged way;
5) and (5) affinity purifying the supernatant to quickly obtain a large amount of rabbit polyclonal antibody products.
2. The method of claim 1, wherein the optimized B lymphocyte culture medium of step 3) is: RPMI1640+ 10% blank rabbit serum + 1% tryptone + CD40L + IL 6.
3. The method of claim 1, wherein the peripheral blood of the rabbit obtained after the third immunization in step 1) is subjected to preparation of a peripheral blood monocyte suspension.
4. The method of claim 3, wherein the peripheral blood mononuclear cell suspension of step 1) is prepared by:
a) adding 20mL of lymphocyte separation solution Ficoll into a 50mL centrifuge tube;
b) taking 10mL of third-immunized anticoagulated rabbit venous blood and 10mL of sterile DPBS according to the ratio of 1:1, fully and uniformly mixing, and slowly overlapping on the layered liquid level along the tube wall by using a pipette;
c) horizontal centrifugation is carried out for 400g multiplied by 30 minutes;
d) sucking peripheral blood mononuclear cells by a pipette and adding the peripheral blood mononuclear cells into a 50ml centrifuge tube;
e) adding DPBS to a constant volume of 45ml, centrifuging for 300g multiplied by 10 minutes, and washing cells twice by using the DPBS under the same condition;
f) after the last centrifugation, the supernatant was discarded, RPMI1640 containing 10% FBS was added, the cells were resuspended, and counted.
5. The method of claim 1, wherein the coated antigen of step 2) is an immunogenic protein, a polypeptide crosslinked to KLH or BSA.
6. The method of claim 5, wherein the step 2) of obtaining antigen-specific B lymphocytes using the antigen-coated high affinity plate comprises:
i) filtering the antigen by using a 0.22 mu m filter element, coating a high affinity plate with the filtered antigen, and coating overnight at 4 ℃;
II) the next day, the supernatant was blotted and washed twice with 10ml PBS, blocked with 5% FBS/PBS, and blocked at room temperature for 2 hours or at 37 ℃ for 30 min;
III) adding the peripheral blood mononuclear cells separated in the step f) in the step 1) into a high affinity plate according to the amount of 5M/plate, and incubating for one and half hours at 37 ℃;
IV) washing the incubated plate with RPMI1640 for 3 times;
v) collecting the separated cells.
7. The method of claim 1, wherein the conditions of the expanding culture of step 4) are: antigen-specific B lymphocytes were cultured in a dish in a 5% CO2 incubator at 37 ℃ for 5 days, then transferred to a conical flask, cultured from the dish for 5 days, then transferred to the conical flask, further cultured in a 5% CO2 shaking incubator at 37 ℃ for 15 days, and the supernatant was collected by centrifugation.
8. The method of claim 1, wherein the affinity purification method of step 5) is: affinity chromatography was performed using an antigen affinity column.
9. A B lymphocyte culture medium, which is characterized in that tryptone is used as a nitrogen source.
10. The application of tryptone in preparing a culture medium for producing rabbit polyclonal antibody by B lymphocyte culture.
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| CN114317432A (en) * | 2021-12-31 | 2022-04-12 | 河南赛诺特生物技术有限公司 | Screening method of rabbit B lymphocytes capable of recognizing multiple antigens and preparation method of multiple rabbit-derived engineered antibodies |
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